Hi,
I am culturing BMDC using B6 mice. After 1 week I harvested the cells and stained with CD11b and CD11c (figure 1). But the double positive cell percentage is only about 40%. Is it normal?
Then I stimulated the cells with LPS for 48h and run the flow again. Why there is a shift of the souble positive cell (red circle)?
Thanks
I think there are a lot of dead cells for naive BMDC, you need to check the culture condition to detect the stressful factor, as
cell density is too high or too low,
incubator temperature and co2 percentage
whether changing culture media during the week or not,
possible dryness during changing the media or harvesting the cell.
Hi,
Like Abdel, I think the events are not good in your initial file. You have look into your cultures.
Also, use a live/dead antibody to get a clear picture about dead cells.
You may also need to use DC enrichment kits from BD or STem cells company, to enrich your cells to get a better population.
Check the culture protocol, may be you need to vary the days of culture.
Best,
Hamid
Just from your SSC and FSC, you have a lot of dead cells for a BMDC culture. Either your isolation for flow is causing something weird, or your culturing conditions are not correct.
Also, what are you gating on in the SSC and FSC in the post-LPS graphs? Why does it say lymphocytes? It's doesn't even look like you are gating on the same cell population. So that shift you are seeing in the double positive is probably due to the gating change in the SSC and FSC. Remember your FI is normalized to the gated cell population in the SSC/FSC. And you need to re-comp your CD11c and CD11b.
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