"Herpes Simplex Virus: Propagation, Quantification

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Herpes Simplex Virus: Propagation, Quantification, and Storage Article  in  Current protocols in microbiology · November 2005 DOI: 10.1002/9780471729259.mc14e01s00 · Source: PubMed

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Herpes Simplex Virus: Propagation, Quantification, and Storage

UNIT 14E.1

Human herpes simplex viruses (HSV) are large, double-stranded DNA viruses that are enveloped and contain structured capsids and a proteineous layer located between the envelope and the capsid referred to as the tegument (Roizman and Knipe, 2001). HSV-1 is a neurotropic herpesvirus that causes a variety of infections in humans. It remains latent in the neurons of its host for life and can be reactivated to cause lesions at or near the initial site of infection. Recurrent infections result from the lytic replication of the virus after reactivation from the latent state. During productive lytic infection in cultured cells, glycoproteins in the viral envelope interact with receptors on the host cell, including heparan sulfate and the herpesvirus entry mediators. HSV-1 gene expression proceeds in a tightly regulated cascade, and changes in its levels during infection are usually the consequence of transcriptional regulation. The first viral genes expressed during infection, termed the immediate-early (IE) genes, are stimulated by incoming virion VP16 and are transcribed in the absence of de novo viral protein synthesis. The IE gene products 0, 4, 22, and 27 function cooperatively to regulate the expression of all classes of viral genes. The early genes are expressed next and encode proteins mainly involved in viral DNA synthesis. The last genes expressed are the late genes, and they mainly encode virion components such as VP16 and glycoproteins. The late genes are further divided into the leaky-late and true-late classes of genes. The true-late genes absolutely require viral DNA synthesis for their production. HSV-1 generally completes its replication cycle within 12 to 18 hr of infection. This unit describes protocols required for the propagation and quantitation of HSV. These include splitting of cultures (see Support Protocol 1), freezing cells (see Support Protocol 2), and thawing cells (see Support Protocol 3), as well as preparing virus stocks (see Basic Protocol 1), determining virus titers (see Basic Protocol 2), and picking virus plaques (see Basic Protocol 3). Additionally, methods are detailed for preparing whole infected cell extracts (see Basic Protocol 4), detecting HSV proteins by separation on DATDacrylamide gels (Basic Protocol 5) and subsequent immunoblotting (see Basic Protocol 6), and indirect immunofluorescence (see Basic Protocol 7). These methods may be used for clinical isolates and laboratory strains of both HSV types 1 and 2. HSV-1 serves as the prototype of the Herpesviridae. There are three main laboratory strains of HSV-1 that are used throughout the world and are available from the ATCC. They are HSV-1(17syn+), HSV-1(F), and HSV-1(KOS). Note that HSV1(17syn+) has been completely sequenced and is available in Genbank (Locus: HE1CG; Accession numbers: X141112, D00317, D00374, S40593). CAUTION: Human herpes virus is a Biosafety Level 2 (BSL-2) pathogen. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information. NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly. NOTE: All culture incubations should be performed in a humidified, 37◦ C, 5% CO2 incubator unless otherwise specified. Animal DNA Viruses Contributed by John A. Blaho, Elise R. Morton, and Jamie C. Yedowitz Current Protocols in Microbiology (2005) 14E.1.1-14E.1.23 C 2005 by John Wiley & Sons, Inc. Copyright "

14E.1.1

BASIC PROTOCOL 1

PREPARING VIRUS STOCKS Before any experiments that require virus can be done, the virus must be properly grown up into a stock, titered, and characterized. There are several ways to make a virus stock, as well as varying opinions on the best way to maintain the virus. Described below is a straightforward method for preparing a virus stock, which may be scaled up accordingly. The virus storage method used here allows for repeated freeze-thawings without significant reduction in virus titers. The virus concentration in a stock is expressed as plaque forming units (pfu) per milliliter. It should be recognized that unless infectious virions are purified by banding in a density gradient (Pomeranz and Blaho, 2000), all stocks will contain noninfectious particles along with the infectious virions.

Materials Cell line of choice (e.g., Vero; see Table 14E.1.1) grown to confluence in 75-cm2 tissue culture flasks (see Support Protocol 1 for culture techniques) 199V medium (see recipe) HSV stock (ATCC), titered (see Basic Protocol 2) DMEM/5% NBCS medium (see recipe) Sterile milk (see recipe) Freezer box, cardboard (optional) 15-ml conical tubes, sterile Probe sonicator (e.g., Branson Sonifier) with microtip Screw-capped cryovials Additional reagents and equipment for titering virus (see Basic Protocol 2) Infect cells with virus 1. Aspirate medium from a confluent 75-cm2 flasks of the appropriate cell type. 2. Prepare 3 ml of 199V medium containing HSV at a multiplicity of infection (MOI) of 0.01 pfu/cell according to the following formula:

0.01 × (2 × 107 ) × (1/titer in pfu/ml) = volume (ml) virus stock needed. Here, 0.01 represents the desired MOI in plaque-forming units (pfu)/cell (see Basic Protocol 2) and 2 × 107 is the approximate number of Vero cells per confluent 75-cm2 flask. For example, if the stock has a titer of 1 × 108 pfu/ml, then one must add 2 µl (0.002 ml) to infect one 75-cm2 flask at an MOI of 0.01. 199V medium contains less serum than cell maintenance medium and is used solely when inoculating cells with virus.

3. Add virus/medium inoculum to cells and incubate 2 hr at 37◦ C to allow cells to absorb virus.

Table 14E.1.1 Examples of Commonly Used HSV-Permissive Cell Lines

HSV: Propagation, Quantification, and Storage

Cell line

ATCC

Growth conditions

Uses

Vero (African Green Monkey Kidney Cells)

#81-CCL

DMEM containing serum, Growing virus stocks and passage every 3–4 days, titering 37◦ C, 5% CO2 Indirect immunofluorescence

HEp-2 (Human Epithelial Carcinoma Cells)

#23-CCL

DMEM containing serum, Preparation of infected cell passaged every 3–4 days, extracts 37◦ C, 5% CO2 Indirect immunofluorescence

14E.1.2 Current Protocols in Microbiology

4. Aspirate 199V and add 3 ml fresh DMEM/5% NBCS medium to flask. 5. Incubate 2 to 3 days, until 100% of the cells display cytopathic effect (CPE). CPE caused by HSV on Vero cells is quite distinct and involves rounding up of the cells off the plate, along with numerous biochemical alterations to the cells themselves (Roizman and Knipe, 2001).

Harvest virus and store virus stock 6. When 100% CPE is observed, tighten the cap of the flask and place it at −80◦ C for at least 15 min. Use of a cardboard freezer box to hold the virus stock simplifies this procedure.

7. Carefully remove the flask from the freezer and carry it into a biosafety hood. Allow the flask to warm up slowly at room temperature in the hood. Loosen and then tighten cap to equilibrate the air pressure. Do not allow the flasks to warm up too quickly or to get knocked around. Be very careful not to break the flask. 8. As the flask warms, add 3 ml sterile milk to a fresh 15-ml sterile tube. The use of milk as a stabilizer is a more effective way of storing HSV, as compared to storage in medium alone. Powdered milk is cheaper than BSA, and it remains stable through the autoclaving procedure. Virus titers have been found to decrease dramatically when stock is stored in medium, while they are significantly retained with milk storage.

9. As the ice in the flask begins to thaw, shake the virus/cell suspension (often referred to as a “herpes-Slurpee”) over the monolayer to resuspend all cellular material in the flask. 10. Remove the cell suspension from flask using a 5-ml pipet and add it to the 15-ml tube containing the sterile milk. 11. Gently resuspend cells in the milk. Maintain the virus on ice from this point on. 12. Sonicate three times, each time for 30 sec using a Branson Sonifier with microtip at an output setting of 4 (timer on “Hold”; % Duty Cycle on “Constant”; approximate Power Output of 15%), letting the cell suspension cool on ice for 1 min between sonications, to free virus particles from the cellular debris. 13. Split virus stock into smaller aliquots and transfer to sterile, screw-capped cryovials. Label stock with virus name, passage, and date, and store at −70◦ C or colder.

Dividing the suspension into aliquots prevents excess freezing and thawing of the virus every time it is removed from the freezer. Label stock by virus name, passage, and date. It is imperative that virus be stored a temperatures of −70◦ C or colder. Storage at higher temperature rapidly leads to loss of infectivity.

DETERMINING VIRUS TITERS Before viruses can be used in experiments, the titer of the virus stock must be determined. The protocol below utilizes the fact that HSV encodes a receptor that binds the Fc portion of human immunoglobulin molecules (Roizman and Knipe, 2001). Addition of pooled human immunoglobulin precludes the need to grow virus under solid supports, such as agar, and greatly facilitates rapid virus titering. This ensures that the observed plaques result strictly from cell-to-cell spreading and not secondary infection by extracellular virus particles.

BASIC PROTOCOL 2

Materials Cell line of choice (e.g., Vero) DMEM/5% FBS medium (see recipe) 199V medium (see recipe)

Animal DNA Viruses

14E.1.3 Current Protocols in Microbiology

Virus stock (see Basic Protocol 1) 20 mg/ml pooled human immunoglobulin (Sigma) in distilled H2 O Phosphate-buffered saline with potassium (KPBS; see recipe) Methanol KaryoMax Giemsa Stain stock solution (Invitrogen) 25-cm2 tissue culture flasks Plugged, sterile pipet tips Fine-point marking pen Inverted microscope Additional reagents and equipment for splitting cells (see Support Protocol 1) Prepare cells and virus dilutions 1. Split Vero cells (Support Protocol 1) into a sufficient number of 25-cm2 tissue culture flasks for titration in duplicate (see step 3) using DMEM/5% FBS such that they will be ∼90% confluent in 24 hr (e.g., split 1:5 for a concentration of ∼4 × 106 cells/25-cm2 flask). The next day, use these cells for titering. The titration is performed in duplicate to ensure accuracy.

2. Add 1 ml 199V medium to each of a series of tubes. Using a sterile pipet, add 10 µl virus stock to the 1 ml of 199V in the first tube and vortex (this is now a 10−2 dilution). Transfer 10 µl from the 10−2 tube to the next tube containing 1 ml of 199V and vortex (this is now a 10−4 dilution). Transfer 100 µl from the 10−4 tube to the next tube and vortex to prepare a 10−5 dilution. Repeat until the 10−8 dilution has been reached. Discard 100 µl from last tube to keep the volumes constant. Make serial dilutions of virus at least 10−6 , 10−7 , and 10−8 . Make sure to use a clean, plugged pipet tip each time. This is crucial. Virus stocks are routinely of very high titer and repeated usage of a pipet tip for dilutions will affect the results.

3. Label each 25-cm2 flask of confluent cells (from step 1) with the dilution of virus that it is to receive. Aspirate the medium. Add 1 ml of each diluted stock to the corresponding flask of cells. 4. To allow virus to absorb, incubate 2 hr with constant gentle rocking, or swirling every 30 min. During the incubation, combine 3.38 µl of 20 mg/ml pooled human immunoglobulin with 9 ml DMEM/5% NBCS medium to prepare a solution containing 7.5 µg/ml human immunoglobulin. 5. Aspirate the virus inoculum from each flask and add 3 ml of the DMEM/5% NBCS containing pooled human immunoglobulin prepared in step 4. 6. Incubate ∼2 days, until plaques are visible.

Stain plaque dishes The following steps need not be performed under sterile conditions. 7. Aspirate medium from dishes. Rinse each monolayer twice with KPBS. 8. Add 1 ml methanol to each dish and leave flat for 5 min at room temperature to fix cells. HSV: Propagation, Quantification, and Storage

9. Dilute the KaryoMax Giemsa Stain stock solution 1:10 with distilled water. Aspirate the methanol and add 2 ml of the diluted Giemsa stain to each flask. Incubate flat for 20 min at room temperature.

14E.1.4 Current Protocols in Microbiology

10. Discard the stain down a sink. Rinse cells gently with cold tap water until the liquid is clear. Dry inverted flask on a paper towel. 11. Count plaques (circle or mark each with a fine-point marking pen) viewing through an inverted light microscope at low (10×) magnification. While a phase-contrast microscope is not neccesary, it may help some investigators to more easily distinguish plaques. To determine titer of the stock, average the duplicate number of plaques counted for a given dilution. This is the number before the exponent. The dilution is the exponent. For example, if four plaques are counted on a 10−8 dish, the titer is 4 × 108 . Since this is an infectious center assay, titers are expressed as pfu/ml.

PICKING VIRUS PLAQUES FOR PLAQUE PURIFICATION To insure that the virus stock is not a mixture of different viruses, it is necessary to plaquepurify the isolate. Often, after a virus has been grown up, the stock needs to be tested for contamination. The protocol below provides a rapid method for screening several virus isolates by picking plaques. Once the plaques are picked, subsequent “plaque-pure” virus stocks are made. These should be used for characterizing the virus (see below).

BASIC PROTOCOL 3

Materials Virus stock (see Basic Protocol 1) DMEM/5% NBCS medium (see recipe) 199V medium (see recipe) 20 mg/ml pooled human immunoglobulin (Sigma) in distilled H2 O Sterile milk (see recipe) 1% (w/v) agarose in KPBS (see recipe for KPBS; store at 4◦ C) Phosphate-buffered saline containing potassium (KPBS; see recipe), sterile 25-cm2 tissue culture flasks Plugged, sterile pipet tips Fine-point marking pen 15-ml snap-cap tubes Bent Pasteur pipet (see recipe) Hand-held battery-operated pipetting device (e.g., Pipet-Aid; Drummond Scientific) Probe sonicator (e.g., Branson Sonifier) with microtip Additional reagents and equipment for splitting cells (see Support Protocol 1) Isolate plaques 1. Split Vero cells (Support Protocol 1) appropriately into 25-cm2 tissue culture flasks such that they will be 90% confluent in 24 hr (see Basic Protocol 2, step 1). 2. To ensure that the plaques will be spread out in the dish, dilute the stock down to its estimated titer by making serial dilutions of virus stock in 199V medium in the same fashion as for virus titering (see Basic Protocol 2, step 2). Only ∼20 to 50 plaques per dish are desirable.

3. Aspirate the medium from each flask of confluent cells prepared in step 1, replace with 1 ml diluted virus, and incubate 2 hr at 37◦ C to allow the virus to absorb, swirling every 30 min. During the incubation, combine 1.125 µl of 20 mg/ml pooled human immunoglobulin with 3 ml DMEM/5% NBCS medium to prepare a solution containing 7.5 µg/ml human immunoglobulin. Animal DNA Viruses

14E.1.5 Current Protocols in Microbiology

4. Aspirate medium from each flask and add 3 ml of the DMEM/5% NBCS containing pooled human immunoglobulin prepared in step 3. Incubate at 37◦ C until plaques are seen (usually within 2 days). 5. Choose and circle 5 to 10 plaques, preferably ones with space between them, using a fine-point marking pen. As noted below (see Commentary), HSV infection of monolayer cells yields distinctive phenotypes, including enlargement and rounding of cells. Bear in mind that certain mutant viruses will have plaque phenotypes that differ slightly from that of wild-type virus.

6. Label one 15-ml snap-cap tube per plaque to be picked, including plaque number, virus, date, and any other appropriate parameters. Add 2 ml 199V and 2 ml sterile milk to each tube. 7. Heat 1% agarose in KPBS in a microwave until agarose is completely dissolved. When the mixture is cool enough to touch (∼45◦ C), aspirate the medium from each dish (step 4) and add 3 ml of the 1% agarose in KPBS. 8. After the agarose has hardened (∼2 to 5 min), pick plaques using a bent Pasteur pipet in a hand-held battery-operated pipetting device (e.g., Pipet-Aid) by sucking up the agarose above the plaque and transferring it to the corresponding tube prepared in step 6, pipetting up and down to release the agar plug. Make sure that it enters the tube. 9. Freeze plaque stocks at −80◦ C for ≥15 min.

Prepare high-titer virus stocks 10. Thaw plaque stocks from step 9 on ice. Sonicate on ice three times, each time for 10 sec using a Branson Sonifier with microtip at an output setting of 4 (timer on “Hold”; % Duty Cycle on “Constant”; approximate Power Output of 15%), letting the virus suspension cool on ice for 10 sec in between sonications. Use equivalent settings if a different sonicator is used.

11. Use 0.5 ml sonicated plaque stock to infect a 90% confluent 25-cm2 flask of Vero cells (prepared as in step 1). Incubate 2 hr at 37◦ C to allow the virus to absorb, swirling every 30 min. 12. Wash monolayer by first aspirating off the milk and then rinsing with 2 ml prewarmed sterile KPBS. 13. Aspirate KPBS and add 3 ml DMEM/5% NBCS medium. Incubate until nearly 100% CPE is observed. In order to obtain good yields, it is very important that the cells go as far along in the infection as possible. This can take up to 2 to 3 days.

14. When 100% CPE has been achieved, freeze flasks at −80◦ C for ≥15 min.

15. Thaw flasks in biological safety hood (work with no more than three to four flasks at a time). Swirl the virus/cell suspension (often referred to as “herpes-Slurpee”) over the bottom of the flask to resuspend all cellular material. 16. When all of the ice is thawed, transfer the medium to a 15-ml tube containing 3 ml sterile milk. Mix by pipetting up and down a few times and place the tubes on ice. Label tubes with appropriate parameters.

HSV: Propagation, Quantification, and Storage

14E.1.6

17. Sonicate on ice three times, each time for 30 sec using the same instrument and settings as described in step 10, letting the virus suspension cool on ice for 30 sec in between sonications. 18. Store at −80◦ C or use ∼0.5 ml directly for infections. Current Protocols in Microbiology

SPLITTING CELLS Both Vero and HEp-2 cells are adherent cell lines that can be grown in almost any type of culture vessel. Covered dishes must always be kept in a CO2 incubator. Capped (filterless) flasks (75-cm2 or 25-cm2 ) do not require such an incubator but require the injection of filtered CO2 prior to tightening; these gassed flasks may be placed in any 37◦ C environment, including water baths.

SUPPORT PROTOCOL 1

Note that it is important to consider the relative confluency of the cell monolayer prior to splitting. Cells should be split once they reach between 95% to 100% confluency. The doubling times of some cell lines are significantly longer than other, standard lines. Therefore, such cells should be maintained at a higher cell density (e.g., never going below 60% confluency).

Materials DMEM/5% FBS medium (see recipe) Cells: Vero or HEp-2 (Table 14E.1.1) Phosphate-buffered saline with potassium (KPBS; see recipe), sterile 70% ethanol Trypsin-EDTA (Invitrogen; also see recipe) Culture vessels: 75- or 25-cm2 tissue culture flasks Prepare flasks 1. Prewarm DMEM/5% FBS medium and sterile KPBS in a 37◦ C water bath for 10 to 15 min. In general, DMEM with fetal bovine serum (FBS) is used for maintain cells for passaging, whereas DMEM with newborn calf serum (NBCS) is used for cells to be infected with virus.

2. Before use, wipe down all bottles (medium, KPBS, and trypsin-EDTA), aspirator, pipetting devices, and biological safety hood with 70% ethanol. 3. Using sterile technique, transfer 10 ml DMEM/5% FBS medium into each new 75-cm2 flasks into which the cells are to be split or transfer 3 ml of the medium into each new 25-cm2 flask into which the cells will be split. If the cell line being passed contains a plasmid encoding a particular gene of interest, it will often also contain a gene encoding a protein for resistance to a particular drug or antibiotic. The presence of this drug ensures that only the cells containing the plasmid will divide. Thus, this antibiotic should be added to fresh culture medium prior to splitting cells.

Trypsinize cells 4. Remove the cells to be split from the incubator. Tilt flask so that medium pools in the corner of the flask away from the cell monolayer. Using a sterile Pasteur pipet, vacuum aspirate the medium at the corner such that the pipet tip does not come into contact with the cells. 5. Rinse cells with 5 ml sterile KPBS, being sure to wash entire surface in order to remove all serum-containing medium. Aspirate the KPBS with a sterile Pasteur pipet at the corner opposite the cells. 6. Repeat the wash described in steps 4 and 5. 7. Add 2 ml trypsin-EDTA and rock the flask back and forth, ensuring that all of the cells are coated. Remove the trypsin-EDTA quickly (within 30 sec) using a sterile Pasteur pipet.

Animal DNA Viruses

14E.1.7 Current Protocols in Microbiology

8. Tighten the cap of the flask and incubate in 37◦ C incubator for 2 to 5 min. It is not necessary to place flasks in the incubator after applying trypsin, but doing so will speed up the process of detaching cells. Some cell lines are more sensitive to trypsin. Overtrypsinizing cells can lead to a loss of cell-surface factors from which the cells cannot recover. Some cells even require a rinsing step to remove any residual enzyme that is left behind. To do this, resuspend the detached cells in KPBS, centrifuge several minutes at 800 × g using a tabletop clinical centrifuge, and resuspend pellet in cell culture medium.

9. Tap flask to dislodge cells and check under microscope to make sure most cells are detached and floating. 10. Using sterile technique, add an appropriate quantity of prewarmed DMEM/5% FBS to the trypsinized flask, according to the desired split. For example, if splitting 1:4, add 4 ml to the cells at this step.

Split cultures 11. Using a 5-ml pipet, triturate the cells ∼30 times by pipetting up and down, squirting the medium against the bottom of the flasks each time. Transfer the cells to the 75or 25-cm2 flasks according to the desired split. For the 1:4 example above, transfer 1 ml of the cell/medium mixture to the medium that was transferred to the labeled flask in step 3.

12. Put the flask on its side and rock it gently to fill the bottom surface of the flask with the cells. 13. Place the flask in the incubator and continue incubating. Loosen the cap of the flask if using a CO2 incubator. SUPPORT PROTOCOL 2

FREEZING CELLS In order to maintain proper quality control, it is recommended that cells not be passaged more than 20 to 30 times. This ensures that they will always be at approximately the same passage during infection experiments, to help maintain reproducibility and consistency. It is therefore necessary to freeze the cell stocks in aliquots that can be rethawed as needed. As a general rule of thumb, it is suggested that each time a new stock is thawed, it be one of the older aliquots. Upon thawing, the cells should be expanded and then a portion frozen, before two to three passages, for future use.

Materials Phosphate-buffered saline with potassium (KPBS; see recipe), sterile DMEM/5% NBCS medium (see recipe) Dimethylsulfoxide (DMSO), sterile 70% ethanol Confluent flasks of Vero or HEp-2 cells (see Support Protocol 1) Trypsin-EDTA (Invitrogen; also see recipe) Liquid nitrogen Tabletop clinical centrifuge 1.5-ml cryovials Liquid nitrogen tank or freezer Prepare CFM and rinse cells 1. Prewarm sterile KPBS in a 37◦ C water bath for 10 to 15 min. HSV: Propagation, Quantification, and Storage

2. Depending on the number of flasks that are to be frozen, combine DMEM/5% NBCS medium and DMSO as described in Table 14E.1.2 to prepare an appropriate amount of cell freezing medium (CFM). Place CFM on ice.

14E.1.8 Current Protocols in Microbiology

Table 14E.1.2 Recipes for CFM Based on Number of Flasks Used

Number of flasks

Amount of DMEM/5% NBCS (ml)

Amount of DMSO (µl)

2

2.7

300

3

3.6

400

4

4.5

500

5

5.4

600

3. Before use, wipe down all bottles (i.e., medium, KPBS, and trypsin-EDTA), aspirator, pipetting devices, and biosafety hood with 70% ethanol. 4. Remove the cells that are going to be frozen from the 37◦ C incubator. Using a sterile Pasteur pipet, aspirate the medium at the corner opposite the cell monolayer. 5. Rinse cell layer with 5 ml prewarmed sterile KPBS (step 1). Be sure to wash entire surface in order to remove all serum-containing medium. 6. Using a sterile pipet, aspirate the KPBS with a sterile Pasteur pipet at the corner opposite the cells 7. Repeat steps 5 and 6 for an additional wash.

Trypsinize cells 8. Add 2 ml trypsin-EDTA and rock the flask back and forth, ensuring that all of the cells are coated. 9. Remove the trypsin-EDTA quickly (within 30 sec) using a sterile Pasteur pipet. 10. Tighten the cap of the flask and incubate in 37◦ C incubator for 2 to 5 min. 11. Tap the sides of the flask to dislodge cells. 12. Resuspend cells in 2 ml DMEM/5% NBCS and transfer to a 5-ml tube. From this point forward, keep cells on ice.

Freeze cells 13. Centrifuge cells 3 to 4 min at 800 × g, 4◦ C, using a tabletop clinical centrifuge, and resuspend cell pellets in CFM at ∼1 × 107 cells/ml.

14. Distribute cells in CFM evenly between desired number of cryovials (∼750 µl/ cryovial).

15. Transfer cells to a −80◦ C freezer as soon as possible and leave there overnight or up to 2 weeks to allow cells to equilibrate, then transfer to −150◦ C in a liquid nitrogen tank or liquid nitrogen freezer for long-term storage.

THAWING CELLS Cells permissive for HSV are available from the ATCC and are provided as DMSOcontaining frozen stocks which must be properly thawed to ensure recovery of viable cells. Choosing the type of cell in which to grow virus depends on the purpose of the infection. Certain cells are highly permissive for virus replication and are therefore used to prepare virus stocks. Others are more suited for the characterization of virus replication. Table 14E.1.1 presents some examples of commonly used cell lines. Note that the conditions outlined below are suitable for Vero and HEp-2 cell lines, which are commonly used; however, media, culture vessel, optimal pH, and CO2 concentrations may vary depending on the cell line being thawed.

SUPPORT PROTOCOL 3

Animal DNA Viruses

14E.1.9 Current Protocols in Microbiology

Materials DMEM/5% FBS medium with antibiotics (see recipe) Cryovial containing frozen cells (see Support Protocol 2, or purchase from ATCC), maintained at −70◦ C or below 70% ethanol 75-cm2 flask (or alternative culture vessel) Inverted tissue culture microscope 1. Prewarm DMEM/5% FBS medium at 37◦ C for 15 to 20 min. 2. Prepare a 75-cm2 flask containing 10 to 12 ml of the prewarmed culture medium adjusted to optimal pH with 5% CO2 /95% air. 3. Remove vial containing cells from the freezer and immerse in a 37◦ C water bath. Agitate so that cells thaw rapidly (within 1 min). 4. Wash outside of vial thoroughly with sufficient 70% ethanol to sterilize (∼5 ml). 5. In a sterile hood, transfer thawed cells to the flask from step 2. Pipet up and down several times to ensure that all cells are distributed throughout the medium. 6. Place flask in 37◦ C incubator overnight. 7. On the following day, replace old medium (containing DMSO from the freezing medium) with fresh prewarmed DMEM/5% FBS medium. 8. Monitor cell growth using inverted microscope. Passage cells (see Support Protocol 1) when they reach confluency (cell foci are touching each other). BASIC PROTOCOL 4

PREPARING WHOLE INFECTED CELL EXTRACTS FOR IMMUNOBLOTTING One of the easiest and most reliable methods for documenting viral infection is to assay for immune reactivity against specific viral proteins. Numerous antibodies are now commercially available that recognize individual viral gene products. The protocol below, in conjunction with Basic Protocols 5 and 6, is a simple, straightforward method that can be used with almost any antibody raised against an HSV antigen. Determining protein concentrations allows for equal loading of protein on denaturing gels (see Basic Protocol 5). Without equal loading, comparisons cannot be made on the basis of protein accumulations. This method uses a modified Bradford assay provided by BioRad. It requires generating a standard curve using a known stock of bovine serum albumin (BSA). The slope of the graph of BSA concentration versus absorbance at 595 nm (OD595 ) is the calibration factor. Measure protein concentration of each sample as described in step 17.

Materials

HSV: Propagation, Quantification, and Storage

DMEM/5% FBS medium (see recipe) 199V medium (see recipe) Phosphate-buffered saline with potassium (KPBS; see recipe), 4◦ C Buffer A with protease inhibitors (see recipe), 4◦ C Bio-Rad Protein Assay solution Platform rocker Cell scraper: preferably 12 in. (∼30 cm) long 6-ml tubes

14E.1.10 Current Protocols in Microbiology

Tabletop clinical centrifuge Probe sonicator (e.g., Branson Sonifier) with microtip Spectrophotometer Additional reagents and equipment for splitting cells (see Support Protocol 1), infecting cells (see Basic Protocol 1) Infect cells 1. Split cells (see Support Protocol 1) into 25-cm2 flasks using DMEM/5% FBS medium. Incubate cells overnight at 37◦ C/5% CO2 to 95% to 100% confluency. 2. Aspirate medium. Infect confluent cells at an MOI of 5 to 10 in 1 ml prewarmed 199V medium (see Basic Protocol 1, step 2). Always keep the virus on ice and return the vials to −80◦ C as soon as the virus has been used. This will prevent a drop in virus titer upon refreezing.

3. Place flasks on a platform rocker and incubate 1 hr at 37◦ C to allow virus to absorb. 4. Aspirate virus and add 2 ml DMEM/5% NBCS to each flask. Incubate until it is time to harvest. HSV infections are usually completed between 18 to 24 hr post-infection (hpi). To perform a time course of infection, simply stop the infection at each hour post-infection (Pomeranz and Blaho, 1999).

Prepare whole infected cell extracts 5. To harvest, remove flasks from incubator and carefully begin scraping cells off the side of the flask using a cell scraper. Transfer cells and medium to a 6-ml tube on ice. 6. When all samples have been harvested into tubes, gently centrifuge for 5 min at 3500 rpm in a tabletop clinical centrifuge at 4◦ C. 7. Carefully aspirate supernatant and discard. Resuspend cells in 1 ml ice-cold KPBS and transfer samples to correspondingly labeled 1.5-ml microcentrifuge tubes. 8. When all samples have been transferred, microcentrifuge 10 to 15 sec at maximum speed, 4◦ C. Carefully aspirate supernatant and discard. 9. Gently resuspend cells in 300 µl of ice-cold Buffer A containing protease inhibitors. 10. Sonicate samples three times, each time for 10 sec using a Bronson Sonifier with microtip at an output setting of 2, letting the extract cool on ice for 10 sec between sonications. Always keep samples on ice while sonicating.

Determining infected cell protein concentrations 11. Perform a standard Bradford assay (also see APPENDIX 3A) using the following reaction: 1590 µl H2 O 400 µl Bio-Rad Protein Assay solution 10 µl protein extract (substitute 10 µl Buffer A to prepare blank). Measure absorbance in a spectrophotometer at 595 nm. 12. Proceed directly to gel electrophoresis (see Basic Protocol 5) followed by immunoblotting (see Basic Protocol 6) or store samples in aliquots up to 1 to 2 years at −80◦ C. The extracts should not be thawed and refrozen more than two times. Store in aliquots to ensure extract integrity.

Animal DNA Viruses

14E.1.11 Current Protocols in Microbiology

BASIC PROTOCOL 5

PREPARING AND RUNNING DATD-ACRYLAMIDE GELS WITH HSV WHOLE INFECTED CELL EXTRACTS FOR IMMUNOBLOTTING Denaturing gel electrophoresis (SDS-PAGE) is one of the most convenient assays for assessing HSV infection. It is preferable to use polyacrylamide gels that are cross-linked with N,N' -diallyltartardiamide (DATD). DATD has many advantages over bisacrylamide, as it allows fine resolution of post-translationally modified viral polypeptides, including glycoproteins (Brown and MacLean, 1998).

Materials Mild laboratory detergent 70% ethanol Petroleum jelly 1.4 µg/ml ammonium persulfate Protein gel solutions A, B, and C (see recipe) 20% (w/v) SDS (APPENDIX 2A) TEMED 30% acrylamide/bisacrylamide (see recipe) 1× protein running buffer (see recipe for 10×) Whole infected cell extract (see Basic Protocol 4) Buffer A (see recipe) 4× disruption buffer (see recipe) Gel-forming apparatus: Glass plates for 20 × 20–cm gel 2-mm spacers Combs Large binder clips Syringes Vertical electrophoresis apparatus, power supply, and cables Bent needles for removing bubbles from electrophoresis chamber Boiling water bath Pour the gel The gel must be made before the extract samples are prepared for loading. 1. Thoroughly wash glass gel plates with a mild detergent. Rinse, dry thoroughly, and wipe down with 70% ethanol. 2. Using a syringe, outline the larger glass plate with a thin line of petroleum jelly along both sides and the bottom. There are numerous gel systems available. This method guarantees gels that never leak.

3. Place spacers on top of the petroleum on the glass and adjust into place. Add a thin line of petroleum jelly on top of the spacers and carefully place the other glass on top. Using large binder clips, clip both sides and the bottom to prevent leaks. 4. Stand plates up vertically. Using the appropriate-sized comb, mark on the plate at ∼1 cm from where the wells will end.

5. Prepare the separator gel in a 125-ml flask, according to the percentage of the gel desired, typically (for 20 × 20 gel with 2-mm spacers):

HSV: Propagation, Quantification, and Storage

4.8 ml H2 O 15 ml 1.4 µg/ml ammonium persulfate 7.5 ml protein gel solution A 3.16 ml protein gel solution C

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300 µl 20% (w/v) SDS 18 µl TEMED. The percentage of the gel used should be determined by what protein the researcher is trying to identify. If the researcher is looking for a low-molecular-weight protein, a higher percentage gel should be used.

6. Upon mixing the above ingredients, immediately pour the gel, using a 10-ml pipet, up to the line marked with the comb (see step 4; approximately the bottom three-quarters of the gel). The dimensions of the separator gel will be needed when detecting HSV proteins by immunoblotting (see Basic Protocol 6).

7. Gently add a layer of water on top the separating gel using a Pasteur pipet and allow the separator gel to polymerize. 8. When polymerized, remove the top layer of water using a syringe. Tilt the glass plates until all of the water is removed. 9. Prepare the stacking gel (top 1/4 of the gel) in a 50-ml centrifuge tube, according to the percentage of the gel desired, typically (for 20 × 20 gel with 2-mm spacers):

1.61 ml H2 O 3 ml 1.4 µg/ml ammonium persulfate 750 µl protein gel solution B 638 µl 30% acrylamide/bisacrylamide 2 µl TEMED.

10. Immediately pour the stacking gel using a 5-ml pipet. Put the comb into place and wait for the stacking gel to polymerize completely. 11. Remove the clips and bottom spacer. Use a Kimwipe remove any excess petroleum jelly. Failure to remove excess petroleum jelly may cause the gel not to run properly.

12. Stand the gel vertically and align so smaller plate and comb face inward. Clip plates onto a vertical electrophoresis apparatus. 13. Add 1× protein running buffer to the top and bottom chambers. Make sure that the top does not leak. 14. Remove the bubbles between the two plates in the bottom chamber with a curved (bent) needle on a syringe. 15. Mark the lanes on the glass plates. 16. Remove the comb and rinse the wells with buffer. Samples can now be loaded onto the gel.

Load samples on gel 17. Calculate the volumes of protein extract needed to ensure that there is 50 µg of extract protein loaded in each well. Add buffer A to the whole infected cell extract sample mixtures to make bring all samples to equal volumes. 18. Add 4× disruption buffer to each sample for a final concentration of 1×. Boil samples in a water bath for 5 min, then microcentrifuge 2 to 3 sec at maximum speed to pellet insoluble material. 19. Load samples onto the gel.

Animal DNA Viruses

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Perform electrophoresis 20. Run the gel at a constant current of 70 mA. Running a 2-mm 15% DATD-acrylamide gel at 70 mA typically takes 4 to 5 hr.

21. Perform immunoblot staining (see Basic Protocol 6). BASIC PROTOCOL 6

DETECTION OF HSV PROTEINS BY IMMUNOBLOTTING This method, performed on the gel that was run in Basic Protocol 5, utilizes an electrical transfer method in a tank apparatus, as this is the technique that yields the most efficient transfer of high-molecular-weight polypeptides. While other transfer methods are available, e.g., semi-dry and vacuum-based ones, these techniques suffer from low transfer efficienies. This method also utilizes an alkaline phosphatase-conjugated secondary antibody system, which is ideal for visualizing viral proteins. Chemiluminescence methods may also be used, but these are prone to high background levels due inherent crossreactivites of the available anti-HSV primary antibodies.

Materials Gel containing separated HSV proteins (see Basic Protocol 5) Transfer buffer with and without SDS (see recipes) 0.1% Ponceau S (optional; see recipe) Phosphate-buffered saline (KPBS; see recipe) 5% milk/KPBS (see recipe) Tris-buffered saline (TBS; see recipe) Primary antibodies: HSV-specific antibodies are available from the Rumbaugh-Goodwin Institute for Cancer Research (http://www.rgicr.org/) Secondary antibody, alkaline phosphatase–conjugated (Sigma or Fisher); fluorescently tagged antibodies may be obtained from Molecular Probes 1% BSA/KPBS (see recipe) Tris-buffered saline with Tween 20 (TBST; see recipe) AP buffer (see recipe) 15 mg/ml nitroblue tetrazolium chloride (NBT) in 70% dimethylformamide (DMF) 30 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in 100% DMF Whatman no. 1 filter paper 0.45-µm nitrocellulose transfer membrane Plastic dish large enough to accommodate gel Flat glass plate slightly larger than gel Electroblotting apparatus: tank transfer system including transfer cassette and power supply (e.g., Bio-Rad) Prepare the transfer stack 1. Measure the dimensions of the separator gel. Cut two pieces of Whatman no. 1 filter paper to a slightly larger size, i.e., allowing an extra centimeter on each side. Avoid wasting nitrocellulose and filter paper by being accurate in gel dimensions.

2. Cut a piece of 0.45-µm nitrocellulose transfer membrane according to the dimensions measured. Be careful in handling; nitrocellulose is delicate.

HSV: Propagation, Quantification, and Storage

3. Add transfer buffer without SDS to a plastic dish and place a flat glass plate slightly larger than the cut filter paper in the buffer. 4. Write “top” on one piece of filter paper. Wet the nitrocellulose and filter paper in transfer buffer without SDS.

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5. Place the bottom piece of filter paper on the glass plate, followed by the nitrocellulose. Center and smooth to make sure there are no air bubbles trapped under the paper. 6. At this point, turn off the power supply to the protein gel. Discard the protein running buffer in the top chamber. Unclip the gel and remove it from the chamber. Place the gel flat on a tabletop lined with paper towels. Slide out the side combs and gently separate the two plates using a metal spatula. 7. Using a razor blade, carefully remove the stacking gel, slide it onto a paper towel, and discard. Remove any gel below the blue buffer front line and slide it onto a paper towel to discard. Cut a corner of the gel for orientation. 8. Roll the gel off the glass plates using a razor blade, allowing it to fall onto the nitrocellulose. Gently move the gel into place (congruent with nitrocellulose membrane). 9. Put the second, “top” piece of filter paper onto the gel to form a “sandwich.” Smooth out any air bubbles. Remove the sandwich from the buffer.

Perform the transfer 10. Place the sandwich into an appropriate transfer cassette. The top filter paper should be facing the black terminal (cathode) side of the cassette. Orientation of the gel is very important when transferring. If the “top” is not facing the black side and/or the cassette is placed in the transfer apparatus incorrectly, the gel will be transferred the wrong way and the proteins will be lost.

11. Assemble and close the transfer cassette. Place cold transfer buffer with SDS into the transfer apparatus rig. Insert the cassette into the rig nearest to the anode (red side), oriented so that the “top” side of the cassette faces the black pole of the apparatus (cathode). 12. Plug into power supply and run at 100 V in a cold room (4◦ C). It will take ∼2 hr to completely transfer a 2-mm, 15% acrylamide gel.

13. Turn off the power. Take apart the cassette. Rinse cassette, sponges, and rig with tap water to remove salts before storing. Depending upon time available, the researcher may opt to air dry the nitrocellulose membrane or go directly to next step and block in 5% milk. The membrane may also be stained at this point using 0.1% Ponceau S (see recipe), which allows for visualization of almost all the proteins on the membrane. This gives the researcher the ability to see whether there are any air bubbles or if there was a problem with the transfer. This step is not necessary, but it is advised.

Probe for immune reactivity of viral proteins 14. If the blot was dried, rewet it completely in KPBS. Block the membrane by incubating in 5% milk at room temperature for 1 hr or overnight at 4◦ C. Blocking in milk reduces the background binding of antibodies. The blocked membrane may also be air dried and stored.

15. Rinse the blot six times, each time with 10 ml TBS. 16. If it is desirable to cut the blot into sections (e.g., to stain with more than one antibody), carefully cut the blot with a razor blade using the markers as a guide. Loading prestained molecular weight markers onto the gel is advised if one desires to cut the membrane.

17. Dilute primary antibody in 1% BSA/KPBS. It is important to know the required dilution for the primary antibody. A titration of antibody dilutions should be performed to determine the optimal dilution.

Animal DNA Viruses

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18. Add the appropriate primary antibody to each blot and incubate at 4◦ C. Incubation with primary antibodies against viral proteins should take only 2 hr and may be done at room temperature. However, detection of certain cellular proteins requires overnight incubation at 4◦ C.

19. Remove the primary antibody. Rinse the blot three times, each time with 10 ml TBST The antibody stock may be reused and should be stored at 4◦ C.

20. Dilute secondary antibody 1:1000 in 1% BSA/KPBS. Add the diluted secondary antibody to the blot and incubate for exactly 1 hr at room temperature. The secondary antibody must correlate with the primary. For example, if the primary antibody is from mouse, the researcher must make sure to use an anti-mouse secondary antibody. Antibodies should be diluted (e.g., 1:1000) in 1% BSA/KPBS. The authors generally suggest secondary antibodies coupled to alkaline phosphatase.

21. Discard secondary antibody and rinse three times, each time for 15 min with 10 ml TBST. 22. Rinse three times, each time for 5 min with 10 ml TBS. 23. Rinse once with 10 ml AP buffer for 1 to 2 min. 24. Prepare developer solution by diluting 15 mg/ml NBT and 30 mg/ml BCIP together 1:500 in AP buffer. 25. Add developer solution to the blot and wait for the bands to appear. Stop by rinsing with water before the blot becomes over-developed. BASIC PROTOCOL 7

USING INDIRECT IMMUNOFLUORESCENCE TO LOCALIZE VIRAL PROTEINS WITHIN CELLS Indirect immunofluorescence allows the researcher to visualize the localizations of various viral proteins within the infected cell. Refer to Pomeranz and Blaho (1999) for examples using specific antibodies to show that different viral proteins reside in different places in the infected cell. The following is a standard method for confirming productive viral infection.

Materials

HSV: Propagation, Quantification, and Storage

Ethanol Cells of choice, growing in tissue culture DMEM/5% FBS medium (see recipe) Virus stock (see Basic Protocol 1) 199V medium (see recipe) DMEM/5% NBCS medium (see recipe) Phosphate-buffered saline (KPBS; see recipe) 2.5% paraformaldehyde (see recipe) Acetone, −20◦ C 10 µg/ml human immunoglobulin in 1% BSA/KPBS (see recipe for 1% BSA/KPBS) Primary antibody 1% BSA/KPBS (see recipe) Fluorescently conjugated secondary antibody ProLong Antifade Kit (Molecular Probes) Clear nail polish

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25-mm2 coverslips 6-well or 33-mm2 tissue culture dishes Dark plastic box Glass microscope slides Additional reagents and equipment for splitting cells (see Support Protocol 1) Prepare cover slips with cells 1. Dip a coverslip in ethanol and flame to sterilize. Place carefully in dish or well. 2. Seed an appropriate number of the cells of choice (see Support Protocol 1 for splitting technique) in 2 ml DMEM/5% FBS onto the sterilized coverslips in the well/dish to achieve confluency on the next day. Incubate cells overnight.

Infect cells 3. Optional: Perform a “synchronized infection,” by placing dishes on ice for 20 min prior to infection and maintaining them on ice during virus absorption to ensure that all cells are at the same point of the replication cycle throughout infection (Pomeranz and Blaho, 1999). This is especially useful when examining the kinetics of viral protein production.

4. Remove medium and infect cells by adding virus stock at an MOI of 15 in 199V medium. Absorb virus 1 hr at 37◦ C (or on ice for synchronized infection). Always keep the virus on ice and return the virus vials to −80◦ C as soon as virus has been used.

5. Aspirate virus and add 2 ml DMEM/5% NBCS to each well/dish. Incubate 1 to 2 hr. 6. Remove cells from the incubator and aspirate off the medium from the edge of the dish to ensure that the cells are not being removed from the coverslip. The remaining steps need not be performed under sterile conditions.

7. Rinse cells twice, each time with 2 ml KPBS at room temperature, aspirating each time.

Fix cells 8. Add 2 ml of 2.5% paraformaldehyde to each well and incubate at room temperature for 20 min. 9. Aspirate the paraformaldehyde and add 1 ml acetone, −20◦ C, to each well. Place at −20◦ C for 3 to 5 min, then aspirate and rinse cells twice as in step 7.

Perform staining 10. Add 2 ml/well of 10 µg/ml human immunoglobulin diluted in 1% BSA/KPBS and block by incubating 1 hr to overnight at 4◦ C. Treatment with this amount of human immunoglobulin was previously shown to be sufficient to neutralize Fc binding by the viral gE and gI proteins (Pomeranz and Blaho, 1999).

11. Dilute the primary antibody appropriately in 1% BSA/KPBS. This dilution varies depending on the antibody.

12. Remove dishes from the 4◦ C refrigerator and aspirate the blocking buffer. Rinse twice with 2 ml KPBS at room temperature. Animal DNA Viruses

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13. Line the bottom of a dark box with Parafilm. Place 50 µl of primary antibody onto the Parafilm. Remove the coverslip containing the infected cells from the well/dish and very carefully place it cell-side-down on the antibody solution in the dark box. Incubate 1 hr at room temperature. Pay special attention to where the cells are located relative to the coverslip. It is extremely important to know the orientation of the coverslip in relation to being “cell-side-up” or “cell-side down.”

14. Remove the coverslip from the box and place it cell-side up in a fresh well or dish. Rinse twice with KPBS at room temperature, leaving the second rinse in the well or dish. 15. Dilute the secondary antibody in 1% BSA/KPBS. This dilution varies depending on the antibody. For example, FITC-coupled antibodies usually use 1:300, while Texas Red–coupled antibodies use 1:150.

16. Remove the Parafilm lining from the dark box and replace with fresh Parafilm. Place 50 µl of the diluted, fluorescently-conjugated secondary antibody on the Parafilm. 17. Remove the coverslip from the well and very carefully place it cell side-down on the secondary antibody solution in the dark box. Incubate for exactly 45 min at room temperature, making sure that the dark box is closed. 18. Remove the coverslip from the box and place it in a fresh well/dish, cell-side-up. Rinse twice with 2 ml KPBS at room temperature, leaving the second rinse in the well or dish.

Mount coverslip on slide 19. While the secondary antibody is incubating, add 1 ml of Component B from the ProLong Antifade Kit to one of the brown vials from the kit that contain the antifade reagent. The mounting solution thus prepared can be stored up to 1 week at −20◦ C protected from light.

20. Drop ∼5 µl of the mounting solution onto a clean microscope slide. Remove the coverslip from the well or dish and very carefully place it cell-side-down on the mounting solution on the slide. 21. Seal the edges of the coverslip with clear nail polish and allow it to completely dry, keeping the slides in the dark box. Place slides at 4◦ C overnight. Slides will look the best when viewed the following day. Slides are typically viewed at 100× magnification with oil immersion using a fluorescence microscope.

REAGENTS AND SOLUTIONS Use deionized or distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Acrylamide, 30%/bisacrylamide Weigh out 145 g of acrylamide and 5 g bisacrylamide. Add 300 ml water and allow these reagents to go into solution with stirring. Adjust volume to 500 ml with water. Filter through a 0.45-µm membrane. Store up to 1 year at 4◦ C in a glass bottle wrapped with aluminum foil HSV: Propagation, Quantification, and Storage

CAUTION: A dust mask should be worn when weighing out powdered acrylamide. Predissolved liquid acrylamide may be substituted

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AP buffer 10 ml 5 M NaCl 50 ml 1 M Tris·Cl, pH 9.5 (APPENDIX 2A) 2.5 ml 1 M MgCl2 (add 20.33 g of MgCl2 to ∼80 ml H2 O; stir and adjust volume to 100 ml with H2 O; store indefinitely at room temperature) Adjust volume to 500 ml with H2 O Store up to 1 month at room temperature Bent Pasteur pipets (for sucking up plaques) Apply heat near the tip of a Pasteur pipet over a low flame until it bends at a very short 45◦ angle. It is important that the tip of the pipet not be allowed to close during the heating.

BSA, 1% in KPBS Dissolve 1 g BSA in 80 ml KPBS (see recipe). Adjust volume to 100 ml with KPBS. Add 100 µl of 20% (w/v) sodium azide for a final concentration of 0.02%. Store up to 3 months at 4◦ C. Buffer A 5 ml 1 M Tris·Cl, pH 7.5 (APPENDIX 2A) 3 ml 5 M NaCl 1 ml 0.5 M EDTA, pH 8.0 (APPENDIX 2A) 4 ml 10% (v/v) Triton X-100 (Sigma) H2 O to 100 ml Store buffer with above ingredients up to 6 months at room temperature Just prior to use, add the following protease inhibitors to an appropriate-sized aliquot of Buffer A (e.g., 1 ml): 0.1 mM phenylmethylsulfonylfluoride (PMSF; add from 0.1 M stock in methanol; APPENDIX 2A or Sigma) 0.01 mM L-1-chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride (TLCK; add from 0.01 M stock in H2 O; Sigma) 0.01 mM L-1-chlor-3-(4-tosylamido)-4-phenyl-2-butanone (TPCK; add from 0.01 M stock in ethanol; Sigma) Disruption buffer, 4× Combine the following in a 15-ml snap-cap tube: 4 ml 20% SDS (APPENDIX 2A) 2 ml of 1 M 2-mercaptoethanol 2 ml of 1 M Tris·Cl, pH 7.0 (APPENDIX 2A) 2 ml H2 O ∼2 mg of bromophenol blue (Sigma) Store in aliquots up to 3 months at −20◦ C DMEM/5% FBS or DMEM/5% NBCS medium Add 6.25 ml of 10,000 U penicillin/10,000 µg streptomycin (Pen/Strep), 25 ml fetal bovine serum (FBS) or newborn calf serum (NBCS), and 50 µl filter-sterilized 250 mg/ml Fungizone to 500 ml of Dulbecco’s Modified Eagle’s Medium (DMEM). In general, FBS is used to maintain cells to be passsaged while NBCS is used for cells to be infected. While powdered DMEM may be used, premade liquid DMEM is preferred, since it precludes the need for a large-scale filter-sterilization apparatus. Animal DNA Viruses

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Milk in KPBS, 5% Dissolve 5 g nonfat dry milk in