It sounds like you’re doing everything carefully, so I agree—it’s probably not the protocol itself but small details in execution. One of the first things to consider is the thawing process. Even slight differences in how quickly you thaw, how gently you dilute out the DMSO, and how you wash the cells can make a big difference in viability. Rapid thawing in a 37°C water bath followed by immediate dropwise dilution into pre-warmed media is ideal. Centrifuging too harshly or using cold media can stress the cells right at the start. Some people find that resting PBMCs overnight before isolating monocytes improves outcomes—have you tried that?

Monocyte isolation can also be a stress point. Magnetic separation needs to be quick and gentle, and any residual beads left in the culture could affect differentiation. If there’s too much mechanical stress during pipetting or if cells stay at room temperature too long, that might also impact cell health. It’s worth thinking about whether your technique is subtly different from your labmates in these early steps.

Culture conditions are another possible factor. The type of plastic, coating of the plate, and even where the wells are located on the plate can matter. Evaporation from outer wells of a 96-well plate can lead to cell death, especially during long cultures. Using a plate sealer or filling the outer wells with PBS can help stabilize conditions. Also, FBS batch variability can be a hidden issue. Even if you’re using the same lot, it’s worth confirming that.

When you add cytokines, make sure they’re fully reconstituted and filtered to avoid aggregates or contaminants like endotoxin. Media should be pre-warmed and equilibrated to avoid shocking the cells. The cytokine concentrations might also be just fine on paper but behave differently in practice depending on handling.

Donor variation might be contributing more than it seems. Some donors naturally yield monocytes that are harder to differentiate or more prone to apoptosis. Even when using two donors side by side, one might just inherently respond worse to your setup. Unless there’s a known background on the donor, it can be hard to rule out.

Lastly, if your infection procedure happens before you check viability, the infection itself might be contributing to the problem. Timing, MOI, or the prep of the pathogen could influence survival, especially if the cells were already stressed going in.

Many thanks Attapong Sinkitjasub for your time and the detailed feedback, I shall keep that in mind the next time I isolate cells and handle them in general.

Warm regards and Thanks

Vartika

Monocytes don't like freeze thaw cycles, it severly affects their viability in long term culture. The key to better DCs here is to use freshyl isolated monocytes. Additionally cell density is fairly important and should be optimized.

Update: For anyone facing similar problems, I asked a labmate to watch me while isolating monocytes with the CD14+ monocyte isolation kit, and she suggested I do the washing steps more gently, trying to not introduce any bubbles while doing so. It helped a lot and I do not see any dramatic cell death anymore.

Vartika Singh You've hit on a crucial point that I emphasized from our very first discussion: it's all about the small details. The meticulous care you put into every steps-like the precise preparation of dilutions, thorough washing, gentle centrifugation, and delicate mixing-makes a tangible difference.

I'm glad to hear you're able to address those issues effectively. I wish you the best of luck and continued success in your lab work!