Thawing Protocol for Cryopreserved Human PBMC - Cellular ...

Thawing Protocol for Cryopreserved Human PBMC 

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CTL has developed an optimized, serum-free media platform for standardized work with cryopreserved PBMC. Typically, PBMC 

show higher antigen-specific T cell responses over lower background when tested using the CTL serum-free platform. These 

PBMC can be processed and tested in serum-containing media as well, but careful testing of the serum batch used in both the 

wash and test media is recommended in order to avoid serum-mediated mitogenic or suppressive effects. 

The following protocol provides instructions for the handling of the PBMC using the CTL serum-free media platform. 

A. Storage of Cryopreserved PBMC. 

The CTL cryopreserved PBMC from the ePBMC ® library have been mailed to you under conditions that secure their full 

functionality during the shipment. Please store the cryopreserved PBMC vials in liquid nitrogen immediately upon receipt, 

and keep them in liquid nitrogen until the day they will be thawed. Avoid storing at lower temperatures and avoid 

Transient Warming Events during storage. When stored under these conditions, the CTL cryopreserved PBMC will maintain 

full-functionality for several years. 

B. Storage and Preparation of CTL Anti-Aggregate Wash Supplement 20x. 

Cells that die during freezing/thawing release DNA strands that can cause cell clumping and aggregation. Therefore, the 

use of DNAse-containing wash solutions is recommended while thawing PBMC. Moreover, cells show increased metabolic 

activity after thawing as they compensate for the stress reaction — this needs to be accounted for by using nutrient-rich 

wash and test media. CTL offers specifically formulated media for optimal PBMC recovery and functionality after thawing. 

CTL Anti-Aggregate Wash Supplement 20x contains DNAse and essential nutrients in a 20x, ready-to-use formulation. 

Store CTL Anti-Aggregate Wash Supplement 20x solution at -20 o C. Shortly before the intended use, for each vial of PBMC 

to be thawed, thaw 1 vial (1 ml) of CTL Anti-Aggregate Wash Supplement by placing it into a 37 o C water bath and let it 

warm to 37 o C (about 5 min). At the same time place 20x the volume of sterile RPMI into 37 o C water bath. 

For preparing the ready-to-use CTL Anti-Aggregate Wash Solution 20x, add 19+1 (vol/vol) warm RPMI to the 

CTL Anti-Aggregate Wash Supplement 20x. Keep ready-to-use CTL Anti-Aggregate Wash Solution at 37 o C in CO 2 

incubator until use. For best results, use within 1 hour. 

C. Storage and Preparation of CTL-Test Medium 

• CTL-Test Medium has been formulated for the optimal performance of cryopreserved PBMC. 

• CTL-Test Medium is a ready-to-use formulation, with the exception that fresh L-glutamine needs to be supplemented 

prior to use. Store CTL-Test Medium at 4 o C protected from light. Store L-glutamine in small aliquots (1-5ml) at -20 o C. 

On the day of intended use, thaw L-glutamine, and supplement CTL-Test Medium with 1% (vol/vol) freshly thawed 

L-glutamine, e.g., add 1 ml L-glutamine to 100 ml of CTL-Test Medium. CTL-Test Medium supplemented with 

L-glutamine can be stored at 4 o C and protected from light for up to 1 month, however, L-glutamine must be replenished 

every 10 days. 

Please note that the information contained in this document is privileged and confidential and protected from disclosure. 

You are hereby notified that any dissemination, distribution, or copying of this document is strictly prohibited. © 2012. All rights reserved. 

Cellular Technology Limited • 20521 Chagrin Boulevard • Shaker Heights, OH 44122-5350 USA 

+1 216-791-5084 • +1 888-791-4005 Toll Free • +1 216-751-1373 Fax • reagents@immunospot.com • www.immunospot.com


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• L-glutamine supplemented CTL-Test Medium should always be added warm (37 o C) to cells. Ideally, the medium should 

be placed into a CO 2 incubator in a tube with lose cap for 20 minutes to warm up and for the pH to reset itself. (CTL 

media are buffered. Slight fluctuations in pH might occur, resulting in fluctuation of color.) 

• Each PBMC cryovial will yield between 10 and 20 million PBMC. If, e.g., the PBMC are to be plated at 4 million/ml 

(400,000 per well), prepare 5ml of CTL-Test Medium for each vial of PBMC thawed. 

D. Thawing of the cryopreserved PBMC 

• Warm up CTL Anti-Aggregate Wash Solution (20ml ) and CTL-Test Medium (5ml) by placing tubes with lose cap into 

37°C CO 2 incubator for 20 minutes. Both media should be at 37°C and CO 2 equilibrated when added to the cells. Raise 

the temperature in the cryovial that contains the PBMC rapidly to 37°C. It is recommended to use a 37°C sand or glass 

bead bath (available from CTL). Using a 37°C water bath is adequate, but it increases the chance of contamination. 

Under both conditions, the temperature should rise to 37°C in 10 minutes. 

• Aseptically transfer the cells from the cryovial into the 50ml conical tube that has been pre-warmed in a 37°C water 

bath. (At this point, the contents of up to 4 cryovials from the same sample can be pooled.) Pour cells gently into tube 

to minimize shear forces and air bubbles. 

• Rinse each emptied cryovial with 1ml of warm (37°C) CTL Anti-Aggregate Wash Solution to recover residual cells. 

Slowly, drop-by-drop, add the rinse to the cells in the 50ml tube while gently swirling that tube. Pipette the rinse gently 

because sheer forces will induce apoptosis in the cells. Subsequently, add slowly (over 1 minute) more warm (37°C) 

CTL Anti-Aggregate Wash Solution, diluting the content of the cryovial to ~6x of the original volume (e.g., when 

thawing a single vial, final volume should be 10ml). 

• Centrifuge cell suspension at room temperature at 330g for 10 minutes with rapid acceleration and brake on high. 

• Decant the supernatant, and carefully resuspend the cell pellet by tapping the tube (avoid pipetting or Vortexing). Add 

10ml warm (37 o C) thaw solution for each PBMC vial thawed. 

• Count the cells under a UV microscope with Acridine Orange/Ethidium Bromide for the most accurate results 

(counting with Trypan Blue can overestimate cell counts by including apoptotic cells and erythrocytes). Precise counting 

requires that also the numbers of apoptotic cells is established since such cells are still alive, but will be dead before 

the functional assay is completed (inquire with CTL for dyes that permit staining of apoptotic cells for counting by 

UV microscopy. Please be advised that the use of some automatic cell counting devices can lead to a high level of 

inaccuracy in the counting results if they are not calibrated and adjusted in direct reference to manual counting of 

freshly thawed samples. We advise to verify viability first by visual assessment until all the machine generated results 

have been verified. Viability should be over 80% (typically, it’s over 95% for CTL’s ePBMC ® cryopreserved PBMC). 






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• During counting, the PBMC in the CTL Anti-Aggregate Wash Solution should be kept in the CO 2 at 37°C incubator 

with the cap loosened slightly until the cells are ready to be plated. 

• When ready to plate the PBMC, centrifuge them at 330g for 10 minutes at room temperature with rapid acceleration 

and the brake on high. 

• Discard supernatant and resuspend the cell pellet by tapping (avoid shear forces). Adjust to the appropriate 

cell concentration by adding warm (37°C) CTL-Test Medium that has been supplemented with 1% fresh L-Glutamine 

(e.g., for ELISPOT assays to 4 million/ml if 400,000 PBMC are to be plated per well in 100μl). 

Equipment and Reagents 

(Vendors for generic products are interchangeable – asterisk indicates critical, specifically recommended products.) 

EQUIPMENT: VENDOR: CATALOG NUMBER: 

Centrifuge, Allegra 6R Beckman Rotor Gh3.8A ALR6 

Conical Tubes, 50ml, sterile Fisher 14-432-22 

Gloves Fisher 19-048-575A 

Hemocytometer Reichert Bright-Line 02-671-5 

Pipette Aids, Drummond Fisher 13-681-19 

Pipette Tips (200µl) VWR 53508-783 

Serological Pipette, 2ml Fisher 13-678-11C 

Serological Pipette, 5ml Fisher 13-678-11D 

Serological Pipette, 10ml Fisher 13-678-11E 

Variety MicroPipette (20µl) Eppendorf 21-371-6 

Acridine orange (10mg/ml)* Fisher #A3568 

REAGENTS: VENDOR: CATALOG NUMBER: 

CTL-AntiAggregate Wash Cellular Technology Limited, USA CTL-AA-001 (1 ml) or CTL-AA-005 (5 ml) 

Supplement, 20 x (5ml)* 

CTL-Test Medium* Cellular Technology Limited, USA CTLT-005 (500ml) or CTLT-010(100ml) 

Ethidium Bromide (1%) Fisher Biotech BP1302-10 

RPMI-1640 Fisher Biotech BW12-167Q

Thawing Protocol for Cryopreserved Human PBMC - Cellular ...

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