Peripheral blood mononuclear cells (PBMCs) are among the most commonly used human cells in ELISpot and FluoroSpot assays. They can be isolated from blood by density gradient centrifugation using Ficoll-Paque. Once collected, PBMCs may be used immediately or cryopreserved for later use. This makes PBMCs a practical and flexible sample type for immune monitoring studies.
Whole blood can be collected in a range of anticoagulant tubes. Citrate or heparin tubes are often preferred for ELISpot applications based on practical experience. When a larger cell yield is required, PBMCs may also be prepared from a buffy coat, which is the leukocyte-rich fraction remaining after plasma and most red blood cells have been removed.
To reduce granulocyte contamination, blood should be processed and PBMCs isolated within 8 hours of collection, regardless of the anticoagulant used or the isolation method selected. Granulocytes can interfere with ELISpot and FluoroSpot performance, so timely handling is important. Before beginning the procedure, bring all reagents to room temperature and work under aseptic conditions in compliance with local requirements for handling biological materials.
For whole blood, dilute the sample 1:1 with PBS, such as 10 mL blood with 10 mL PBS. Buffy coat samples are more concentrated and should typically be diluted about threefold. Prepare centrifuge tubes with Ficoll-Paque, using 5 mL in a 15 mL tube or 15 mL in a 50 mL tube. Carefully layer the diluted blood over the Ficoll-Paque without mixing the layers. Centrifuge at 400 × g for 30 minutes at room temperature without the brake. After centrifugation, a whitish mononuclear cell layer will form at the interface above the Ficoll-Paque. Collect this layer carefully with a Pasteur pipette and transfer it to a clean tube. If needed, remove some of the plasma first to make collection easier. To avoid overloading, do not combine cells from more than two Ficoll tubes in a single new tube, and keep the volume below half of the tube capacity.
Wash the collected cells by filling the tube with PBS or serum-free medium and centrifuging at 400 × g for 10 minutes. Discard the supernatant, gently resuspend the pellet, and repeat the wash once more under the same conditions. After the second wash, remove the supernatant, resuspend the pellet in serum-free medium, record the volume, and take a small sample for cell counting. A final centrifugation at 400 × g for 10 minutes can then be performed. Some residual red blood cells or granulocytes may still remain after Ficoll separation. If necessary, these can be removed using commercially available red blood cell lysis buffers or granulocyte depletion reagents.
If the cells will be used immediately, discard the supernatant and resuspend the pellet in the appropriate culture medium with gentle pipetting. The PBMCs are then ready for downstream immunoassays such as ELISpot, FluoroSpot, or flow cytometry.
Use Cellstore PBMC Cryopreservation Medium, a ready-to-use, GMP-manufactured, chemically defined, and protein-free cryopreservation solution designed specifically for PBMCs from peripheral blood, cord blood, and buffy coats. This medium supports high cell density and excellent post-thaw viability.
Before starting, label the cryotubes and prepare a freezing container. Cells should not remain in the cryopreservation medium at room temperature for prolonged periods, as this can reduce viability. Optionally, autologous plasma, serum, or human serum albumin (HSA) can be added according to experimental needs to further enhance post-thaw recovery.
After the final wash and cell counting from the isolation step, centrifuge the cells at 500 × g for 5 minutes at room temperature and remove the supernatant. Resuspend the cell pellet in Cellstore PBMC Cryopreservation Medium to achieve a cell density of 1 × 10⁶ to 1 × 10⁸ cells/mL. A recommended freezing volume is 0.5–1.5 mL per cryotube. A pre-freeze viability of over 90% is preferred for best results.
Gently resuspend the cells to maintain a uniform suspension and aliquot into cryotubes. Place the cryotubes into a freezing container and immediately transfer them to a -80 °C freezer for controlled cooling.
Keep the cryotubes at -80 °C for at least 12 hours. For long-term storage, transfer them to a -150 °C freezer or liquid nitrogen tank.
For thawing, prepare cell culture medium consisting of RPMI 1640 with 2 mM L-glutamine, 10% FCS, 10 mM HEPES, 100 µg/mL penicillin, and 100 µg/mL streptomycin. Also prepare sterile centrifuge tubes, pipettes, a room-temperature centrifuge, a 37 °C water bath, and a 37 °C CO₂ incubator. Before starting, pre-warm the medium needed for the first thawing step. Medium for later washes may remain at room temperature. Remove the cryotube from storage and thaw it quickly in a 37 °C water bath until only a small ice crystal remains. If multiple tubes are being thawed, process them in small batches.
Because DMSO is toxic to cells, the thawed sample should be moved to the next step as soon as possible. Add 0.5 to 1 mL of pre-warmed culture medium slowly to the cryotube, gently resuspend the cells, and transfer the contents into a 15 mL tube. Rinse the cryotube with another 1 mL of medium and add that rinse to the same tube, then fill the tube with additional culture medium. Centrifuge at 300 × g for 10 minutes, remove the supernatant, loosen the pellet by tapping the tube, and resuspend the cells in 1 mL of medium by gentle pipetting. Add more medium to bring the total volume to 15 mL, then centrifuge again at 300 × g for 10 minutes.
After the second spin, discard the supernatant, resuspend the pellet in 1 mL of medium, and add more medium as needed to reach the desired concentration, depending on the expected yield. Incubate the cells in a 37 °C CO₂ incubator for 1 hour to overnight with the cap slightly open. This resting period is also a convenient time to prepare and block ELISpot or FluoroSpot plates.
After resting, gently mix the cells and allow debris or aggregates to settle for about one minute. Carefully transfer the cell suspension, avoiding the debris, into a fresh 15 mL tube. It is best to limit this to no more than two cryotubes per 15 mL tube, or roughly 30 to 40 million cells. Finally, count the cells and assess viability using an automated counter or trypan blue staining under a microscope. Since only viable cells contribute to the assay signal, dead cells and, if possible, apoptotic cells should be excluded from the final count.
Process blood samples and isolate PBMCs within 8 hours of collection to limit granulocyte contamination. During freezing, avoid leaving cells in freezing medium at room temperature for extended periods. When thawing, proceed quickly after the ice has melted to reduce DMSO exposure.
Gently resuspend cells at each step by tapping the tube and pipetting slowly. During thawing and resting, allow debris to settle before transferring the cell suspension to a new tube. This helps prevent clumping and reduces carry-over of dead cells and aggregates.
Use a controlled cooling method with a freezing container, store cryotubes under proper long-term conditions, and provide a sufficient recovery period (1 hour to overnight) in a CO₂ incubator after thawing. Always count viable cells only and adjust concentrations based on actual recovery to ensure optimal performance in downstream assays.
