Prion strain discrimination in cell culture: The cell panel assay Sukhvir P. Mahal*, Christopher A. Baker*, Cheryl A. Demczyk*, Emery W. Smith*, Christian Julius†, and Charles Weissmann*‡ *Department of Infectology, Scripps Florida, 5353 Parkside Drive, Jupiter, FL 33458; and †Institute of Neuropathology, University Hospital of Zurich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland Contributed by Charles Weissmann, October 24, 2007 (sent for review February 5, 2007)
Prions are thought to consist mainly or entirely of misfolded PrP, a constitutively expressed host protein. Prions associated with the same PrP sequence may occur in the form of different strains; the strain phenotype is believed to be encoded by the conformation of the PrP. Some cell lines can be persistently infected by prions and, interestingly, show preference for certain strains. We report that a cloned murine neuroblastoma cell population, N2a-PK1, is highly heterogeneous in regard to its susceptibility to RML and 22L prions. Remarkably, sibling subclones may show very different relative susceptibilities to the two strains, indicating that the responses can vary independently. We have assembled four cell lines, N2a-PK1, N2a-R33, LD9 and CAD5, which show widely different responses to prion strains RML, 22L, 301C, and Me7, into a panel that allows their discrimination in vitro within 2 weeks, using the standard scrapie cell assay (SSCA). standard scrapie cell assay 兩 infectivity 兩 response index 兩 PrP
T
he ‘‘protein only’’ hypothesis proposes that the prion, the pathogenic agent causing transmissible spongiform encephalopathies, consists mainly or entirely of PrPSc, a conformational variant of the constitutively expressed host glycoprotein PrPC (1). Some forms of PrPSc are partially resistant to limited proteinase K digestion and are designated as rPrPSc, in contrast to proteasesensitive forms, termed sPrPSc (2–5). One of the many remarkable features of prions is the existence of distinct strains, originally characterized by the incubation time and the neuropathology they elicit in a particular host (6). Within the framework of the ‘‘protein only’’ hypothesis, the finding that many different strains can be propagated indefinitely in a host homozygous for the PrP gene (Prnp) demands that the strain-specific properties be enciphered in some feature of the pathogenic PrP other than its amino acid sequence, such as its conformation (7, 8) or its glycosylation pattern (9). Biological identification of a prion strain currently demands characterization of its properties in the mouse bioassay and is consequently slow and labor intensive. We therefore investigated the possibility of distinguishing prion strains in cell culture. We have developed a quantitative, sensitive, accurate, and rapid cell-based assay for prions, the standard scrapie cell assay (SSCA) (10). It is based on the use of a highly prion-susceptible N2a cell line and the identification and quantification of individual, rPrPSccontaining cells using automated imaging equipment. The N2a subclone selected for the purpose, N2a-PK1 (PK1 for short), is susceptible to the murine prion strains RML and 22L, but not to Me7, 22A, mouse-passaged 263K, the BSE-derived 301C, or to a variety of prions propagated in species other than the mouse (10). Selective prion susceptibility of cell lines has been reported for PC12 cells (11), GT1-7 cells (12), N2a subclones (13), NIH 3T3 and L929 cells (14), and others (15). In this article, we document the heterogeneity of a cloned cell population in regard to its response to different prion strains. We find that subclones of a cloned PK1 cell population have very different susceptibilities to the RML and 22L strains and that these susceptibilities can vary independently. Importantly, we have established a panel of four cell lines, PK1, CAD5, LD9, and N2a-R33 20908 –20913 兩 PNAS 兩 December 26, 2007 兩 vol. 104 兩 no. 52
(R33 for short), which allows discrimination of the prion strains RML, Me7, 301C, and 22L by the SSCA. Results PK1 Cell Populations Are Heterogeneous in Regard to Their Response to Prion Exposure. To determine prion infectivity by the standard
scrapie cell assay (SSCA), susceptible cells are exposed to an appropriate dilution of the prion preparation, the cells are propagated for three splits to dilute out the original inoculum and allow spreading of the infection in the cell population, and finally 20,000 cells are filtered off onto a membrane and the number of PrPScpositive cells is determined by an ELISA (10). Fig. 1a shows plots of positive cells (from various cell lines, described below) as a function of the logarithm of the dilution of an RML prion sample. The proportion of positive cells reflects the titer of the prion preparation, the susceptibility of the cell population to infection, the extent of lateral spread of infection during cell propagation, and also the level of rPrPSc accumulation in infected cells. We arbitrarily define response index300/3 (RI300/3, or, in this article, RI for short) of a cell population as the reciprocal of the dilution of a standard prion-infected brain homogenate that yields 300 spots per 20,000 cells in the SSCA after the third split. We have previously reported on the selection of a clone of murine neuroblastoma cells highly responsive to the RML prion strain, PK1, as well as of a clone largely resistant, R33 (10). As shown in Fig. 1a, R33 cells were ⬎103 times less responsive to RML than PK1, as judged by the fact that to generate the same number of spots (100) in the SSCA, R33 cells would require a ⬎103 higher concentration of RML brain homogenate than PK1 cells. To assess the homogeneity of PK1 cell populations in regard to their response to prion exposure, we picked at random a set of subclones (designated as CAB series) and determined their RIs to RML and 22L prions (Fig. 2a). Remarkably, sister clones showed not only vastly differing RIs to RML (from ⬍0.1 ⫻ 104 to 130 ⫻ 104) and to 22L (0.29 ⫻ 104 to 120 ⫻ 104) (Table 1), but had very different ratios of responsiveness to the two prion strains, with RI22L/RIRML values ranging ⬎30-fold, from 0.6 to ⬎20 (Table 1). This experiment not only demonstrated the heterogeneity of the N2a cell population with regard to prion responsiveness but led to the unexpected finding that responsiveness to different prion strains varies independently. Moreover, the RIs of individual cloned populations changed rapidly in the course of propagation. In the experiment of Fig. 2b, a single clone was picked and expanded to a population of ⬇105 cells, of which 552 were cloned and challenged with RML and 22L prions. The scattergram (Fig. 2b) again shows Author contributions: S.P.M., C.A.B., and C.W. designed research; S.P.M., C.A.B., C.A.D., and E.W.S. performed research; S.P.M., C.A.B., E.W.S., and C.W. analyzed data; C.J. contributed new reagents; and C.W. wrote the paper. The authors declare no conflict of interest. Freely available online through the PNAS open access option. ‡To
whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/ 0710054104/DC1. © 2007 by The National Academy of Sciences of the USA
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Fig. 1. Responsiveness of PK1, CAD5, LD9, and R33 cells to various prion strain preparations. The cells indicated in the figure (PK1, blue; CAD5, red; LD9, violet; R33, green) were infected with prion preparations and subjected to the SSCA. The cells were infected with a serial 1:3 dilution of 0.1% homogenates of brains infected with RML (a), 22L (b), Me7 (c), and 301C (d) prions. Response Index300,3 (RI300,3 or RI for short) of a cell line for a prion strain is defined as the reciprocal of the dilution required to yield 300 scrapie-positive cells per 20,000 cells after the third split. The characteristic metric adopted in this article is the ratio of the RI of a cell line relative to that given by LD9 cells.
PrPC Levels and Growth Rates of Cell Populations with Different Responsiveness to Prion Strains. It has been suggested that the
degree of susceptibility to prion infection could depend on the cellular PrPC level (12); if so, the dependence might vary for different prion strains. We determined cell surface and total PrPC levels of PK1, R33, and various CAB cell populations by flow cytometry of intact cells labeled with anti-PrP antibody and by Western blot analysis of cell lysates, respectively (Table 1). As shown in the scattergram of Fig. 3a, there is little correlation between total PrP and RI (RML) or RI (22L) with R2 ⫽ 0.12 and 0.05 in both cases; the R2 values for cell surface PrP versus RI (RML) and RI (22L) were 0.17 and 0.23, respectively [supporting information (SI) Fig. 4]. It has been noted that, if the growth rate of cells exceeds that of prions, then persistent infection of a cell population is not sustainMahal et al.
able (16). We therefore determined the growth rate of the CAB subclones (Table 1); as shown in the scattergrams of Fig. 3 c and d, there is little correlation for RI (RML) versus doubling time (R2 ⫽ 0.21) but a more distinct trend for RI (22L) (R2 ⫽ 0.37). Doubling time may be one of several factors contributing to responsiveness. Western blot analysis did not reveal any striking differences in the glycoform pattern and band mobilities of 12 N2a-derived clones (SI Fig. 5). A Panel of Cell Lines with Distinct Responsiveness to Four Prion Strains. In searching for further cell lines susceptible to persistent
infection by prions, we observed that Cath.a-differentiated (CAD) cells (17) were responsive to RML, 22L and 79A prions. The repeated subcloning procedure described earlier (10), challenging with RML, yielded the CAD-2A2D5 line (CAD5 for short), which proved to be responsive to Me7 and 301C as well. It allows quantification of RML at concentrations down to ⬇60 LD50 units/ml. The murine fibroblast cell line L929 was early on found to be responsive to the Chandler scrapie strain (18). It could be persistently infected by Me7 and 22L, and to a lesser degree by RML prions (14). We derived the L929-D9 line (LD9 for short) from L929 cells, selecting for responsiveness to RML as described above. PNAS 兩 December 26, 2007 兩 vol. 104 兩 no. 52 兩 20909
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great variability in the spot numbers determined for the individual subclones challenged with RML and 22L prions. Because the parental clone had been expanded to ⬇100,000 cells for assaying, the variability arose within ⬇17 cell doublings. A clone of the second generation was subcloned similarly and a wide spread of responsiveness was again observed; only a few subclones were more, and most less responsive to RML than the parental clone. (Fig. 2c).
Fig. 2. Responsiveness of PK1 subclones to 22L and RML prions. (a) Sixteen clones derived from the PK1 population (CAB clones, filled circles), as well as PK1 (open triangle) and R33 (open circle) were expanded to confluence in wells of a 96-well plate, and 20,000 cells of each were challenged with 10⫺5 RML and 22L-infected brain homogenates, respectively. The SSCA was performed for three splits as described. The spot numbers are plotted against each other. Two of the PK1 subclones are marginally more responsive to both prion strains than the original PK1 population whereas the remainder lost responsiveness to both strains to varying degrees. (b) One clone from a (encircled) was used to generate a second generation of 552 subclones, which were challenged with a 10⫺4 dilution of RML or 22L-infected brain homogenate. The responses of the parental clone are depicted with a star. (c) Third-generation subclones (264) were isolated from one clone from b (encircled) and also challenged with 10⫺4 dilutions of RML or 22L brain homogenate. Again, the responses of the parental clone from b are shown as a star in c. The axis labels in c refer to all graphs.
LD9 cells proved to be about equally responsive to Me7, 22L, and RML but resistant to 301C (Fig. 1 and Table 2). The four selected cell lines, PK1, R33, CAD5, and LD9, were exposed to serial dilutions of RML, 22L, 301C, and Me7 brain homogenates and subjected to the SSCA for three splits. The proportion of rPrPSc-positive cells (‘‘spots per 20,000 cells’’) was plotted against the logarithm of the dilutions of the four brain homogenates. The linear part of each plot is centered at ⬇300 spots. Fig. 1a shows that the dilutions of RML brain homogenate required to give 300 spots were 5.3 ⫻ 10⫺7 on CAD cells, 8.2 ⫻ 10⫺7 on PK1 cells, and 5.3 ⫻ 10⫺6 on LD9 cells; the cognate RIs are therefore 1.9 ⫻ 106, 1.2 ⫻ 106, and 1.9 ⫻ 105, respectively. Even at the lowest dilution of RML brain homogenate used, 10⫺3, R33 cells failed to
yield 300 spots and the RI was therefore ⬍103. Fig. 1 b–d shows the corresponding plots and RI values for 22L, Me7, and 301C brain homogenates, respectively. Because distinct prion preparations may contain different levels of prions, it is necessary to refer the RI to some other infectivityrelated parameter. We arbitrarily chose as reference value the RI given by the prion preparation in question on LD9 cells. By the criterion of the RI ratio, CAD cells are 9.9, PK1 cells 6.4 and R33 cells ⬍⬍0.005 times as responsive to RML as LD9 cells. Table 2 summarizes the average RIs and RI ratios obtained in nine independent cell panel assays, using the same brain homogenates throughout and distinct aliquots of the frozen-down cell lines; it shows that there is an acceptably low standard error of the mean
Table 1. Response indices, PrP levels, and doubling times of R33, PK1, and PK1 subclones
a b c d e f g h i j k l m
Clones
RI (RML) ⫻ 10⫺4
RI (22L) ⫻ 10⫺4
RI (22L)/ RI (RML)
Surface PrP (% of PK1)
Total PrP (% of PK1)
Doubling time, h ⫾ SD (n ⫽ 3)
PK1-CAB4 PK1-CAB19 PK1-CAB31 PK1-CAB29 PK1 PK1-CAB1 PK1-CAB33 PK1-CAB11 PK1-CAB15 PK1-CAB27 PK1-CAB3 PK1-CAB35 R33
130 83 76 68 46 36 19 7.3 1.9 1.0 0.12 ⬍0.10 ⬍0.10
100 74 95 63 28 120 15 12 20 2.8 0.29 2.0 4.7
0.77 0.89 1.2 0.93 0.61 3.3 0.79 1.6 10.5 2.8 2.4 ⬎20 ⬎47
96 ⫾ 11 53 ⫾ 35 85 ⫾ 30 93 ⫾ 13 100 77 ⫾ 4.0 110 ⫾ 5.7 110 ⫾ 18 82 ⫾ 2.9 110 ⫾ 15 100 ⫾ 8.7 180 ⫾ 120 70 ⫾ 6.8
55 ⫾ 21 85 ⫾ 9.8 76 ⫾ 2.7 85 ⫾ 5.2 100 49 ⫾ 8.6 140 ⫾ 33 75 ⫾ 7.2 80 ⫾ 12 58 ⫾ 4.4 43 ⫾ 1.4 85 ⫾ 13 47 ⫾ 1.5
26 ⫾ 1.6 27 ⫾ 2.2 30 ⫾ 2.7 24 ⫾ 1.3 24 ⫾ 1.1 25 ⫾ 0.50 23 ⫾ 1.0 24 ⫾ 0.50 25 ⫾ 1.3 24 ⫾ 2.2 22 ⫾ 0.90 24 ⫾ 3.3 25 ⫾ 2.9
Clones with the designation ⬙CAB⬙ are sibling subclones derived from the PK1 population. PK1 cells were seeded in 96-well plates at ⬇1 cell per well. Wells in which single colonies developed were identified, and after 10 days the clones were resuspended and expanded for approximately eight additional doublings to generate enough cells for frozen stocks and assays. RIs to RML or 22L scrapie were determined after three splits by the SSCA. PrP levels and doubling times were measured as described in Materials and Methods. 20910 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0710054104
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Fig. 3. Correlation between response indexes and total PrP or doubling time. The data are from Table 1. (a and b) Total PrP versus the RIs for RML and 22L. (c and d) Doubling times versus the RIs for RML and 22L, respectively, of R33, PK1, and various PK1-derived CAB clones. The lowercase letter next to the points refers to the clones listed in Table 1.
associated with the measurements carried out in the course of 15 months. Because there may be some variability in the response of the cell lines it is important to always run a set of standard strains along with unknown prion sample when comparing RI values. Fig. 1 and Table 2 show that each of the four strains presents a distinctive response pattern on the panel of the four cell lines: 22L is propagated on all four cell lines, RML on CAD5, LD9 and PK1, Me7 on CAD5 and LD9 but 301C only on CAD5. There is thus a hierarchy as regards the ‘‘virulence’’ of the strains, 22L ⬎ RML ⬎ Me7 ⬎ 301C; and conversely, a hierarchy in the susceptibility of the cell lines, CAD5 ⬎ LD9 ⬎ PK1 ⬎ R33. Discussion Some cell lines, when exposed to prions, go through a short-lived period in which they produce rPrPSc (it is not known whether infectivity is generated in parallel), designated ‘‘acute infection’’ and then, depending on the strain, either lose the accumulated rPrPSc or proceed to a persistent or chronic state of infection, characterized by continuous propagation of rPrPSc and infectivity (19). Traditionally, susceptibility to a prion strain is equated with
the ability of a cell line to sustain persistent infection. It is not clear whether acute and persistent infection reflect qualitatively different processes (19) or whether acute infection not followed by chronic infection reflects a situation where prion replication proceeds efficiently immediately after infection but at a rate lower than that of cell division, leading to a diluting-out of prions, whereas chronic infection comes about when prion replication holds pace with cell population growth (16). In the SSCA, cells are exposed to prions and then propagated for three passages, during which rPrPSc particles from the inoculum are diluted out and ‘‘acute infection,’’ if any, abates. Also during this period, particularly in the case of PK1 and CAD5 cells, infection spreads through the growing cell population, increasing the proportion of rPrPSc-positive cells to a degree that may be celldependent and to some extent prion strain-dependent. Because the SSCA measures not only susceptibility of a cell population to infection but also the spread of infection under the conditions of the assay, we designate the outcome as ‘‘response’’ of a cell line to a prion strain, and define the ‘‘response index’’ (RI) as the reciprocal of the dilution that gives a designated proportion of infected cells under defined assay conditions. The RI is of course also dependent on the concentration of prions in the inoculum, and therefore, to compare different prion preparations, it needs to be related to a relevant reference value such as the LD50 titer as determined in the mouse bioassay or the rPrPSc concentration of the sample. Because bioassays are time-consuming and because the ratio of infectivity to rPrPSc in various tissues and at various times after infection may be variable, we express RI values relative to the RI on LD9 cells, to obtain an ‘‘RI ratio.’’ Using this metric, we find that RML, Me7, 301C, and 22L give very different and characteristic values on our cell panel, allowing clear distinction of these strains far more rapidly than by the traditional characterization in mice. Because the properties of the individual cell lines may vary to some extent within the same lab, and likely more so from laboratory to laboratory, it is important to run standard strain preparations in parallel with each panel assay. By accruing further cell lines to the panel, a more comprehensive cell-based strain typing system can likely be achieved. Prion-susceptible cell populations are very heterogeneous in regard to their response to prion exposure (10, 13, 20, 21), as highlighted by our finding that the RI of PK1 subclones for a particular prion strain may vary by 3 or more logs (Table 1 and Fig. 2). We have found a similar heterogeneity among CAD5 and LD9 subclones (S.P.M. and C.A.D, unpublished results). We had expected that for one cell line this parameter would vary in concert for different prion strains and were surprised to find that in the case of N2a-derived clones the susceptibility to RML varied not only widely but to a considerable extent independently of susceptibility to 22L.
RI 关(1/dilution) ⫻ 10⫺5兴 Prion 22LBRAIN RMLBRAIN Me7BRAIN 301CBRAIN
RI ratio
CAD5
LD9
PK1
R33
CAD5/LD9
PK1/LD9
R33/LD9
17 ⴞ 4.6 29 ⴞ 5.2 0.12 ⴞ 0.033 0.39 ⴞ 0.054
8.6 ⴞ 2.8 2.8 ⴞ 0.94 2.2 ⴞ 0.74 ⬍⬍0.01
8.1 ⴞ 1.8 14 ⴞ 3.0 ⬍0.017 ⫾ 0.004 ⬍⬍0.01
0.46 ⴞ 0.11 ⬍0.01 ⬍0.014 ⫾ 0.004 ⬍⬍0.01
2.4 ⴞ 0.4 19 ⴞ 7.7 0.1 ⴞ 0.04 ⬎⬎40
1.2 ⴞ 0.17 9.2 ⴞ 3 ⬍⬍0.013 n.d.
0.08 ⴞ 0.014 ⬍⬍0.007 ⬍⬍0.013 n.d.
The Response Index300,3 (RI300,3 or RI for short) is defined as the reciprocal of the dilution of the prion sample that results in 300 spots after the third split of the SSCA. Values ⫾ SEM given for RML, Me7, and 22L are the averages of nine independent assays, carried out over the course of 15 months. RI ratios were calculated for each experiment and then averaged. Values for 301C are from three recent experiments. Because the RI depends on the prion concentration in the sample, this variability is circumvented by expressing the ratio of RIs relative to those given by one cell line, arbitrarily chosen to be LD9. The table shows that these values differ characteristically for the three prion strains propagated in brain. RI ⬍ 0.01, response at 10⫺3 dilution did not reach 300 spots; RI ⬍⬍0.01, no significant response at 10⫺3 dilution. n.d., cannot be determined. The significant values are bold.
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Table 2. Average RIs and RI ratios from nine cell panel assays
How can cell lines, all of which carry the Prnpa allele, distinguish between prion strains that all originated in Prnpa mice? The question also bears on the in vivo selectivity of prion strains for specific brain regions (22–26). In principle, discrimination could occur either at the level of uptake and intracellular trafficking, for example by proteins that recognize different PrPSc structures (including glycans), or at the level of replication, as postulated by the ‘‘conformational selection’’ hypothesis (27). Because at least some features of strain specificity are maintained in cell-free rPrPSc propagation (28–31) (J. Castilla, R. Morales, P. Saa´, and C. Soto, personal communication) and because uptake of rPrPSc seems to be unspecific (32, 33), strain discrimination at the cell surface seems unlikely. All cell lines of our panel contain the Prnpa allele with only the wild type sequence (S. Browning and C.A.B, unpublished results). If translation is faithful and the primary sequence of PrPC is the only determinant of conformational space, these cell lines should be able to propagate all prions stemming from Prnpa donors; however, they do not. Therefore, within the framework of the ‘‘conformational space’’ hypothesis, some cell-specific determinant must modulate the capacity of PrPC to assume a particular conformation. We may consider several possibilities: (i) The conformation of PrP within a fibril may be constrained by the extent and nature of its glycosylation (9, 34–36) and glycosylation may differ in individual cell lines or brain regions (25, 26) or even with the age of the brain (37). At least 52 different oligosaccharides have been reported for Syrian hamster PrPC (38, 39) and some 60 structures for the rPrPSc of Me7-infected mouse brain (40). The finding that rPrPSc associated with distinct scrapie strains acquires different glycosylation patterns in the same cell line (30, 41) is compatible with this hypothesis; however, the glycosylation profile of rPrPSc associated with a particular strain can vary, depending on the brain region or organ in which it is formed (42–44), arguing against a highly specific role of glycosylation in strain determination. (ii) Cell-specific factors such as proteins, glycosaminoglycans (45–47), or small RNAs (48) may codetermine the conformations PrP can assume and maintain. (iii) It is conceivable that the structure of PrPSc, possibly including its glycans, entails recruitment of specific chaperones required for its replication, so that the capacity of a cell to propagate a particular strain may depend on its capacity to express that chaperone. The chaperone repertoires might differ in different cell compartments (30). (iv) Tanaka et al. (49) have proposed a kinetic model supported by their investigation of ‘‘yeast prions,’’ in which persistent infection depends on various parameters, in particular a cell-dependent fibril-cleaving activity that may act differently on different strains (49). The potent tools that have become available with the advent of protein misfolding cyclic amplification (PMCA) (50–52) and the rapid method of strain identification described in this article will be helpful in distinguishing between these possibilities. Whereas heterogeneity of cell populations in regard to susceptibility to a single prion strain is well documented (10, 13), our study reports the remarkable finding that sibling cell lines may show different relative susceptibilities to two prion strains. Whatever the determinants responsible for the different susceptibilities, their expression is obviously highly variable; whether this variability is due to epigenetic differences in the expression of one or more genes or to reassortment of highly aneuploid chromosome sets remains an open question. Materials and Methods Prion Strains. The RML strain (RML I856-II) was obtained from the Prion Unit, University College London and propagated in CD1 mice to give RML CAB001. The Me7, 301C, and 22L strains were from the TSE Resource Centre, Compton, Newbury, U.K. and were propagated in C57BL mice (Charles River Laboratories, Wilmington, MA). In all cases, mice were inoculated with 30 l of 1% brain homogenate and killed when clinical signs became pronounced, between days 144 and 148 (RML), 133 and 143 (Me7), 152 and 164 (301C), and 143 and 149 (22L). Endpoint titrations of Me7 and 22L were carried out on C57BL and 20912 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0710054104
of RML on CD1 mice and evaluated as described in ref. 53. The titers, in LD50 units/g brain, were 108.75 for RML, 108.26 for Me7, 108.26 for 22L. Brain Homogenates. Frozen brains were homogenized for 10 s in PBS (9 ml per g) using a hand-held Ultramax T18 basic homogenizer (IKA Works Inc., Bloomington, NC) at 20 –25,000 rpm. 301C-infected brain was homogenized by hand with a Kontes All-glass Potter-Elvehjem Tissue Grinder Size 23 (Fisher Scientific) under BSLIII containment conditions. Homogenates were stored in small aliquots at ⫺80°C. Thawed homogenates were rehomogenized by passing through a 28-gauge needle; they were not centrifuged at any stage. Standard Scrapie Cell Assay. The SSCA was essentially performed as described (10, 54). In short, prion-susceptible cells were exposed to a serial dilution of the prion preparation for 3 days and, depending on the cell line, propagated for three 1:7 (PK1 and R33), 1:8 (CAD5) or 1:10 (LD9) splits. After reaching confluence after the second and/or the third split, 20,000 cells were deposited on the membrane of a Multiscreen IP96 well 0.45-m filter plate (Millipore, Danvers, MA). rPrPSc-positive cells were identified by an ELISA and counted by using the Zeiss KS Elispot system as described in ref. 10, except that the primary anti-PrP antibody was anti-PrP monoclonal HuM chimeric antibody D18, 0.33 g/ml, purified from the supernatant of CHO cells (55), a gift from R. A. Williamson, and the secondary antibody was AP-conjugated anti-human IgG (1:5,000, cat no. 9042-04; Southern Biotechnology Associates, Birmingham, AL) (54). Selection of Cell Lines. The isolation of PK1 and R33 cells has been described (10). CAD-2A2D5 (CAD5) cells were derived from Cath.a-differentiated (CAD) cells (17), a gift from Dona Chikaraishi (Duke University, Durham, NC). Cells were seeded at ⬇100 cells per 10-cm dish in 10 ml of OBGS (Optimem; Invitrogen) containing 9% bovine growth serum (BGS; HyClone, Logan, UT), 90 units penicillin/ml, 90 g of streptomycin/ml). Approximately 50 colonies were picked and transferred to a 96-well plate. After 7 days, 20,000 cells were transferred to wells of duplicate 96-well plates. After 16 h, one plate was infected with 0.3 ml of 5 ⫻ 10⫺6 RML-infected brain homogenate (RML for short; ⬇103 LD50 units) in OBGS and subjected to the SSCA (using three 1:8 splits, each after 3– 4 days), whereas the uninfected plate was incubated with OBGS alone. The clone giving the highest spot number (CAD-2A2; 700 spots per 25,000 cells, third split) was expanded and subcloned as above, and 14 clones were challenged with 10⫺7 RML. The most positive clone, CAD-2A2D5 (⬇250 spots per 25,000 cells, third split) was expanded and frozen in 50% BGS, 10% DMSO, and 40% OBGS. LD9 cells were derived from murine L929 fibroblasts (ATCC, Manassas, VA). Cells were seeded at an average of 1 cell per well in ten 96-well plates in EMEM-BGS (Eagle’s minimal essential medium with 9% BGS, 90 units/ml penicillin, 90 g of streptomycin/ml). After 14 days, the cells from 96 subconfluent wells containing one to three colonies were suspended in 0.05% trypsin-EDTA (Sigma) and 20,000 cells were transferred to duplicate 96-well plates. After 16 h, one plate was infected with 0.3 ml of 10⫺5 RML in EMEM-BGS, split 1:10 after 3 days, and thereafter every 2–3 days for altogether three splits, and assayed by the SSCA. Cells corresponding to the most positive well (LD929-1B6; 40 spots per 20,000 cells) were expanded and subcloned as described for the CAD cells. Approximately 200 colonies were plated in duplicate 96-well plates; one set of plates was challenged with 10⫺5 RML brain homogenate and subjected to the SSCA. The most positive clone L929-1B6D9 (LD9 for short; 300 spots per 20,000 cells) was expanded and aliquots were frozen in EMEM-BGS, 5% DMSO. Maintenance of Cells. Cells were maintained in OBGS (PK1, R33, CAD5) or EMEM-BGS (LD9) and split 1:10 at confluence or earlier. After six serial passages, cells were discarded and another frozen aliquot was thawed and expanded for use. When necessary, susceptible clones were reisolated from a fresh culture as described above. Cell Surface PrPC Levels. ⬇106 cells were incubated in flow buffer (FB): (1⫻ PBS, 1% bovine growth serum, 1 mM EDTA) with or without 0.7 g of anti-PrP antibody/ml D18 (55) for 20 min at 4°C. Samples were diluted 1:5 in FB and centrifuged for 5 min at 500 ⫻ g, and the cells were suspended in 8 g/ml Alexa Fluor 488-labeled goat-anti-human IgG (Invitrogen). After 20 min at 4°C, the cells were diluted, pelletted, resuspended in FB, and analyzed on a Becton Dickinson LSRII flow cytometer. To exclude cell aggregates, events were gated on single cells using forward scatter height versus forward scatter width parameters. For each cell line, the average fluorescence intensity in the absence of D18 was subtracted from that in the corresponding D18-containing sample. Exposure of HpL3-4 cells devoid of PrP (56) to D18 antibody had no effect on fluorescence.
Mahal et al.
Total PrPC Levels. Cells (⬇7.5 ⫻ 106/ml) were lysed in PBS containing 50 mM Tris䡠HCl (pH 7.5), 0.5% sodium deoxycholate, 0.1% Triton X-100, and 1 tablet of EDTA-free complete protease inhibitor mixture (Roche Applied Science, Indianapolis, IN) per 50 ml. Samples (0.5–3 g of total protein) were subjected to Western blotting, developed with D18 antibody (0.67 g/ml) and HRPconjugated Sheep Anti-Human IgG (1:5,000; GE Healthcare), and visualized with ECL-plus (GE Healthcare). Films were scanned and analyzed by using ImageJ software (National Institutes of Health; http://rsb.info.nih.gov/ij/). The total densitometric signal for all PrP bands was normalized to the signal given by PK1 cells.
suspended, and fluorescence was determined at 485 nm excitation and 535 nm emission in a Thermo Fluoroskan Ascent plate reader. The number of cells in each well was calculated from a standard curve run in parallel. Doubling times were calculated by fitting an exponential curve to at least four points within the range of 2,000 to 10,000 cells. Results represent the average ⫾ SD for three separate experiments.
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MEDICAL SCIENCES
Cell Growth Rates. Approximately 2,000 cells were seeded per well in 96-well plates. Every 12 h, the medium was replaced with 100 l of Hank’s balanced salt solution (HBSS) containing a 1:1,000 dilution of the CyQuant NF dye reagent (Invitrogen, Carlsbad, CA). After 60 min at 37°C, the cells were
ACKNOWLEDGMENTS. We thank A. Aguzzi for suggesting the use of Cath.adifferentiated (CAD) cells in the SSCA assay, J. Collinge (MRC Prion Unit, London, U.K.) for a sample of RML, and A. Williamson (Scripps Research Institute, La Jolla, CA) for D18 antibody-producing CHO cells. We thank C. Lasmezas for discussions and A. Sherman (Scripps Research Institute, Jupiter, FL) for the production of prion strain-containing brain homogenates, endpoint titrations, and the maintenance of prion-infected clones. This work was supported by grants from The Scripps Research Institute and the Alafi Family Foundation.
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PNAS 兩 December 26, 2007 兩 vol. 104 兩 no. 52 兩 20913
