I'm trying to produce a plaque assay with an Eilat virus and C710 cells but whenever I go to stain it seems like the cells are just washed off the wells and I get nothing. I am using the correct plates for cell culture from ThermoFischer.
Here is my protocol:
Complete media – DMEM 4.5g/L Glucose. Supplement with 1% Tryptose phosphate broth, 0.1%
gentamycin, 10% FBS
2. Dilution media – DMEM. Supplement with 1% FBS and 0.1% Gentamycin
3. Overlay media – 2X MEM supplemented with 10% FBS, 2% Tryptose phosphate broth, 0.2%
gentamycin
4. 2% Tragacanth solution prepared in sterile water
Procedure (for one sample, can be scaled up)
1. Seed a 6-well plate with 2ml/well of cells at a concentration of 1.2x10 6 cells/ml.
2. Approximately 24 hours later (when cells are ~90% confluent), thaw EILV/CHIKV virus sample in
a 37C water bath with shaking until completely thawed. Do not incubate at 37C longer than
needed to completely thaw.
3. Prepare Eppendorf tubes corresponding to the number of dilutions (i.e. 9 tubes)
4. Add 900ul of dilution media to each tube.
5. Prepare 10-fold serial dilutions by adding 100ul of virus to the first tube. Mix well and change
tips between dilutions.
6. Remove plate of cells from incubator and label.
7. Aspirate the medium from each well (do not aspirate media from more than two plates at a
time).
8. Carefully add 200ul of diluted virus to the wall of each well beginning with the most diluted virus and working down to the least diluted. Change tips between plates.
9. Rock plates to evenly disperse inoculum
10. Incubate at 28C for 1 hr. Rock the flask every 15 min to evenly disperse inoculum and prevent
cell desiccation.
11. During incubation, prepare overlay by mixing tragacanth solution with 2x MEM overlay medium at a ratio of 1:1. Prepare 12ml per plate.
12. After incubation, add 2 ml of overlay per well
13. Incubate at 28C for 2.5-3 days
14. Aspirate overlay from each well
15. Gently add ~3ml of 10% formaldehyde and incubate at room temp for at least 1 hour
16. Stain with ~1ml of crystal violet solution for 5 min.
17. Remove stain, rinse with water, and dry plates.
I am SO gentle with these cells and always pipette or disperse liquids on the well side and never on the cells directly.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus