When isolating PBMCs, it's essential to use PBS without Mg²⁺ and Ca²⁺ for washing, especially when working with large volumes of blood or buffy coats. This is because these divalent cations can cause PBMC aggregation, which can lead to reduced cell viability and functionality. To further minimize aggregation, a small amount of EDTA (1 mM) can be added to the washing media. Some researchers also include 2% FBS in their washing media, which can help to reduce cell adhesion and aggregation. However, it's crucial to process the PBMCs quickly, as prolonged standing can lead to re-aggregation.

For cryopreservation, we usually store 20 million cells per mL. To achieve this, you will need to prepare cryopreservation media and ensure a gradual temperature decrease during storage. First, determine the number of vials required based on the PBMC count. Then, transfer the vials to a freezing container and cool them down to 4°C. Next, prepare the freezing media by mixing 9 parts of FBS with 1 part of dimethyl sulfoxide (DMSO). Alternatively, you can use a mixture of 70% cell culture media (RPMI 1640, 10% FBS, 1% P/S, 1% pyruvate, 1% HEPES, 1% Glutamax) and 20% FBS with 10% DMSO. Both cryopreservation media formulations give me acceptable recovery rates after cryopreservation (>90% viability and >80% recovery). It's recommended to bring the prepared cryopreservation media to 4°C before use. Resuspend the PBMC pellet with cold freezing media and transfer 1 mL into the cooled vials and cryo-container. Then, transfer the cryo-container to -80°C overnight and the next morning to LN2.

When thawing the cryopreserved PBMCs, it's essential to prepare cell culture media as mentioned earlier and warm it to room temperature. To prevent temperature shocks, retrieve the PBMC vials in reverse storage order, i.e., from LN2 to -80°C and then to a 37°C water bath. Avoid waiting for the ice in the PBMC vials to completely thaw before transferring them to cell culture media for washing. Finally, keep the cells overnight in culture media with 5% CO₂ and 37°C to allow them to recover and adjust before performing downstream assays the next day.

And hopefully, at the end, you can proceed with your downstream assays. I wish you the best and good luck, Magdalena Mijas .