Hi everyone,

I’m working on a project involving the culture of PBMCs and I’d appreciate your insights. We’ve been staining cells with CTV and activating them using ImmunoCult™ Human CD3/CD28 T Cell Activator. Despite this, we’ve observed that approximately 80% of the cells remain undivided by Day 3, and around 25% by Day 5. These percentages of undivided cells seem higher than expected, and we’re curious if others have seen similar patterns. Are these values typical, or might they indicate an issue with the culture conditions?

We’ve tested both total PBMCs and CD4+ T cells (isolated via negative selection), and the results are consistent across both approaches.

For reference, we’ve attached data showing:

• Undivided cells on Day 3, highlighted in orange (based on CTV staining and marker expression).
• A histogram depicting cell divisions in the CD4+ T cell population.

Here are the culture conditions we’ve been using:

• Cell density: 1 million cells/mL
• Medium: cRPMI (RPMI + 10% FCS + 1% PSG)
• Additives: IL-2 (final 5 ng/mL) and supplemented glutamine (final 2 mM)
• IL-2 and glutamine were added on Day 0 and replenished fresh every other day.

Any insights, advice, or relevant experiences would be greatly appreciated!

Thank you in advance for your help.