PBMC Thawing Protocol

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To reduce the risk of contaminating the cells during thawing, we recommend the use of a CTL Bead Bath™(CTL-BB-001) instead of a water bath. Make sure that the temperature of the Bead Bath (or water bath) is at 37°C. 

Prepare CTL Anti-Aggregate Wash™ Medium

For each vial of PBMC to be thawed, thaw 1 vial of CTL Anti-Aggregate Wash™ Supplement 20x (1ml, CTL-AA-001) by placing in a 37°C CTL Bead Bath™(or water bath) for 10 minutes. Dilute 1:20 by adding 19ml of RPMI-1640. 

For best results, prepare CTL Anti-Aggregate Wash™ Supplement 20x within 1 hour of use. ⦿ Place the medium in a 37°C CO2 incubator with a loose cap for a minimum of 20 minutes.  This allows the pH and the temperature to reset.  

Prepare CTL-Test™ Medium

CTL Test™ Medium is a ready-to-use formulation with the addition of 1 vol % fresh L-glutamine before use. (L-glutamine is unstable at 4°C and needs to be frozen for long-term storage). Thaw L-glutamine and add 1 vol % (e.g., 5ml L-glutamine to 500ml CTL-Test™ Medium). 

For best results, pre-warm the L-glutamine-supplemented CTL Test™ Medium before adding it to the PBMC by placing the medium in a 37°C CO2 incubator with a loose cap for a minimum of 20 minutes. This allows the pH and the temperature to reset itself. The medium should be protected from light while working on the bench top (by wrapping it in aluminum foil). After use, the media should be stored at 4°C. 


  1. Raise the temperature in the cryovial that contains the PBMC rapidly to 37°C by placing it in a CTL Bead Bath™ for 8 minutes. (Using a 37°C water bath is adequate, but it increases the chance of contamination.)
  2. Flip the cryovial twice 180° to resuspend the cells.   
  3. Use 1ml pipette to aspirate all medium from the cryovial, transfer into a 50ml conical tube (make sure the tube is labeled with the sample ID). The contents of up to 4 cryovials from the same sample can be pooled at this point.
  4. To recover the residual cells from the cryovial, pipette 1ml warm (37°C) CTL Anti-Aggregate Wash™ Medium into each cryovial, aspirate it, and add to the rest of the cells.  
  5. Using a 10ml pipette, add warm (37°C) CTL Anti-Aggregate Wash™ Medium to the 50ml tube. The first 3ml should be added slowly, 1ml at a time every 5 seconds, while gently swirling the tube. Add the remaining 5ml of CTL Anti-Aggregate Wash™ Medium more quickly from the pipette. The PBMC are now suspended in ~10ml.
  6. Centrifuge cell suspension at room temperature at 330g for 10 minutes with rapid acceleration and brake on high.
  7. Decant the supernatant and carefully resuspend the cell pellet by tapping the tube (avoid pipetting or vortexing). Add 10ml (37°C) CTL Anti-Aggregate Wash™ Medium. Mix the cells by flipping the tube twice 180° with the cap tightly closed. Take a sample for cell counting.
  8. Centrifuge cell suspension at room temperature at 330g for 10 minutes with rapid acceleration and brake on high.  
  9. ⦿ Once centrifuge stops, decant the supernatant, and resuspend the pellet by tapping the tube. Add warm (37°C) CTL-Test™ Medium adjusting the cells to the concentration as planned for plating into the assay (e.g., adjust to 3 million PBMC per ml if 300,000 PBMC are to be plated in 100μl/well). If cell counts are not yet available by the time the centrifuge stops, resuspend the cells in 1ml warm CTL-Test™ Medium, and add the missing volume once the calculation becomes available.

The above protocol summarizes the ideal thawing conditions as established in Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies,” Cells, 2012. 1:313-324. Ramachandran, et al.).

CTL does not recommend “overnight resting” of ePBMC®, but to test the cells right after thawing (“Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays,” Cells, 2012. 1:409-427. S. Kuerten, et al.)

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