I'm doing a growth curve for cells, but most of them started to die in 2 days after seeding into the 12 well plate. I try to infect them with the virus also, but the replication kinetics are not good. The virus seems not to replicate as good as they should be, which may be due to the dying of cells. I seed 1x10^6 cells/well and I start counting 1 day after. They all are single cell, I'm sure about it, and they grow well in the flask. Can anybody give me advice? Thanks in advance.
Dear Nithipong
What is the cell line? and what virus are you investigating? Cells should not dye. Most likely cause is your CO2 supply may not be adequate. Are the media pink?
I'm working with THP-1 cells and infect them with dengue virus.
I think CO2 is okey because other cells are fine.
THP-1, acute monocytic leukemia cells. If that's correct, it is a suspension type cell culture. ATCC suggested "Subculture when cell concentration reaches 8x10^5 cells/mL. Could that mean your seeding density has already gone over the maximum density limit in order to keep them happy?
Try lower cell density if possible
Quoting from ATCC
"cultures can be established by centrifugation with subsequent resuspension at 2 to 4 x 10^5 viable cells/mL. Subculture when cell concentration reaches 8x10^5 cells/mL.Do not allow the cell concentration to exceed 1 x 10^6 cells/mL."
Some times adding equal volume of "spent media" reduces the transfer shock. Also, please check your tissue culture [microtiter] plate. Did you have problems with other cell lines growing in these plates?
I agree that the cell density maybe just too high at the start. For endothelial cell at confluence for 6-well plate is about 1X10^6. For THP-1 cells the number maybe highre since they do not attach to the bottom but I will consider 10^5 cells maybe more appropriate for your experiments.
This cell seeding density is definitely high for any cells. Density of epithellial cells shall be less than fibroblasts. In general, it can be 50000 to 80000 cells per well. Check viable count before seeding. Optimize between 2 to 5% CO2 atmonsphere. Give medium change after 24 hours. Dead cells will cause toxicity. Try partial replacement with conditioned (already used) medium. If cells are just revived from cold storage, sometimes it may take 1-2 passages to grow smoothly. A combination of these factors should 100% work. Initial viability of cells is very imp. Less seeding is always better than overcrowding. All the best.
-you already answer to the type of cells…
other missing information:
-method to plate them?
-How do you qualify them as dead or alive?
I remembrer some organism just too fragile to be plated dying immediately… but symptoms of dead just visible after 2 days…
I agree with everyone to reduce the cell density, so I tried plating 6x10^5 cells/well, instead. One day after, cell concentration reaches 1x10^6 cells/ml and started to died on day 3. I think I should reduce cell concentration more, but I worry about the infection efficiency of the virus, which may reduce also if the cell density is too low. The tissue culture plate is ok, other cell lines can grow well. I used trypan blue exclusion method to determine cell viability.
You can try to infect the monolayer of cells after 48 hrs when cells in a very receptive state of getting infected.
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