I was trying to check some markers on immune cells after stimulation with cd3/CD28 or LPS.
I started with frozen PBMCs, the viability was great, 97%.
I used RPMI1640+10%FBS+1%P/S to culture PBMCs for 24hrs.
I used ultra low plate, just in case the monocytes attach to the bottom.
I also used poly-polyene plate, to avoid monocytes attach to the bottom.
Then the flow data show very little CD14 monocytes left. Even the unstimulated samples. Very few CD14 cells left.
What could be wrong? Thank you!
Best,
Annie
Salah A. Alshehade
There are a few potential reasons why you may be losing CD14+ monocytes:
- Monocytes can tend to adhere to plastic surfaces, so even using low-attachment plates, some loss may occur over 24 hrs. Using an even more low-adherence plate or coating with serum/BSA may help.
- In culture, monocytes can begin differentiating into macrophages or dendritic cells, downregulating CD14 expression. This process may be accelerated by stimulation. Using shorter culture times could minimize differentiation.
- Some monocyte death is expected in culture over 24 hrs. The death rate may be increased by suboptimal culture conditions. Ensuring optimal cell density, serum levels, gentle handling, etc. can improve viability.
- Downregulation of CD14 - Stimulation via LPS, cytokines, or other factors can induce shedding or downregulation of CD14 on monocytes, making them appear CD14-negative by flow cytometry.
- Selective loss - Certain culture conditions may selectively impact monocyte health and viability vs other PBMCs.
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