Hi experts,
Recently I have been learning to perform XC plaque assay. The experiment was done as these steps:
1. 2 x 10^5/well of XC cells were seeded in 6-well TC treated plate, over night.
2. the next day, remove the culture medium, inoculate the cells with frozen viral stock at different dilutions, undiluted, 10-1 to 10-8. Observe CPE in hourly bases. 4 hours later, the viral medium was discarded and the cell culture medium was replaced with fresh cell culture medium with 10% FBS
3. 48 hours later, the cell culture medium was refreshed.
4. 96 hours later, the cells were fixed with ice cold methanol for a hour and stained with commercialized harris hematoxylin for 30min. cells were then washed with PBS, twice
5. CPE or plaque formation was visualized under convert microscope.
These are what I found:
1. with undiluted viral medium, CPE could be seen as early as 2 hours during inoculation.
2. Some "plaques" could be seen in the well infected with high diluted viral medium, but the diameter of the plaque was quite small, some times i can see there were 3 nucleus gathered in the center. Even with harris hematoxylin staining, the plain plaque was hard to see with nude eye.
3. Some "plaques" could also be seen in the negative control wells.
My questions are:
1. Did i do the experiment right? should I start with more cells or less cells? I wonder the reason I didn't see bigger plaque is because the initial cell density was too low.
2. How many days after the inoculation would be a better time to fix cells for plaque counting?
3. The type of plaque that I saw in the negative control was real plaque by some sort of cross contamination or because of the cells were too many and too old?
Any input would be very much appreciated. I am a new hand in this area. The only way for me to learn it is by reading papers. If you know yours or any other organizations could offer a hand-on training opportunity, please let me know. Thanks a lot.
Best
Julia
- Badges
- Science topic