Nov 18, 2025
Isolation, Cell Counting, and Freezing of PBMCs @ ZeBanC
- Merz, MP1,
- Stege, A2,
- ZeBanC - Zentrale Biobank Charité2
- 1Berlin Institute of Health (BIH) @ Charité;
- 2Charité Universitätsmedizin
- Merz, MP: orcid.org/0000-0003-2085-1980;
- ZeBanC - Zentrale Biobank Charité: RRID:SCR_023495
Protocol Citation: Merz, MP, Stege, A, ZeBanC - Zentrale Biobank Charité 2025. Isolation, Cell Counting, and Freezing of PBMCs @ ZeBanC . protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx486kl8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 04, 2025
Last Modified: November 18, 2025
Protocol Integer ID: 231480
Keywords: PBMCs, ZeBanC, EDTA, Heparin, Citrate, PBMC Isolation, LeucoSep, freezing of pbmc, central biobank charité, pbmc, peripheral blood mononuclear cell, zebanc, determination of concentration, cell
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Abstract
This procedure describes the isolation, determination of concentration, and freezing of PBMCs (Peripheral Blood Mononuclear Cells) from blood (EDTA, heparin, citrate) at the Central Biobank Charité (ZeBanC), unless otherwise specified.
Guidelines
The Central Biobank Charite (ZeBanC) works under the best practice laboratory guidelines and ISO:9001 quality standards.
This protocol in its published form has only been validated on machines and chemicals as described here.
In case of doubt, please feel free to consult attached manufacturers SOPs or reach out to us.
Materials
- Ficoll Paque Plus, 500mL (Cytiva #17-1440-03)
- FCS (Fetal bovine/calf serum) (Sigma-Aldrich #F7524-500ml)
- RPMI (Thermo #31870025)
- Dimethylsulfoxide (DMSO) Cell Culture Grade, 250mL (Sigma-Aldrich #D8418-250ml)
- phosphate buffered saline (PBS) without Ca/Mg, 500mL (GIBCO #605294)
- 0.4% trypan blue solution (Fisher Scientific #15250061)
- Isopropanol (to fill Mr. Frosty)
- LeucoSep tubes (Greiner #227290)
- Polypropylen Tubes ("Falcon Tubes"), 50mL (Falcon #352070)
- 10ml serolog. Pipettes (Sarstedt #86.1254.001)
- 25ml serolog. Pipettes (Sarstedt #86.1685.001)
- cell counting slides (for Countess) (Thermo #C10283)
- Mr. Frosty (Thermo #5100-0001)
Equipment
Countess 3 FL Automated Cell Counter
NAME
Automated Cell Counter
TYPE
Thermofisher scientific
BRAND
AMQAF2000
SKU
LINK
Equipment
Megafuge ST1 Plus MD
NAME
centrifuge
TYPE
Megafuge ST1 Plus Centrifuge
BRAND
LINK
Equipment
Safe 2020
NAME
bio safety cabinet
TYPE
Herasafe
BRAND
LINK
class II A2
SPECIFICATIONS
Troubleshooting
Safety warnings
Cell separation medium (Ficoll) and trypan blue solution are cytotoxic.
Be aware that this SOP has NOT been tested for BD Vacutainer‱ CPT‱ Mononuclear Cell Preparation Tubes.
Ethics statement
The Central Biobank Charité (ZeBanC) acts as a core facility/service provider for researchers. When appropriate, the ZeBanC requests to see ethical approval and informed consent statements before biospecimen processing.
Before start
Time to isolation:
Ideally, PBMCs are isolated and frozen within 4 hours of blood collection. However, it is possible to process PBMCs up to 24 hours after collection (during this time some cells will be lost to apoptosis).
In this case, whole blood should be stored in the original tube (heparin, EDTA or citrate) at room temperature (approx. 18-22°C) until isolation.
Things to prepare before starting
1h
Prepare LeuoSep Tubes
- Fill LeucoSep tubes (incl. divider disc) with 15 mL of Ficoll (each) under the bench and seal them (with lid).
- Centrifuge at 1000 x g for 30 seconds (at room temperature) so that all the Ficoll locates below the divider disc.
- If any Ficoll remains above the divider disc, carefully remove it (under the bench) with a pipette and discard it.
- Store filled LeucoSep tubes (closed) at room temperature in a dark place and use within 4–6 weeks.
5m
Heat-incativation [HI] of FCS
- Inactivate FCS in a water bath at 56°C for 30 minutes, stirring occasionally.
- Then portion and freeze (-20°C). Label the bottle/tube/aliq. with [HI] and the freezing date.
- After thawing, store at 4°C and use within 4 weeks.
45m
Prepare and aliquot freezing medium 1 and 2
Freezing medium 1 (consisting of 40% FCS [heat-inactivated] + 60% RPMI)
o For 15 mL stocks= 5 mL FCS + 7.5 mL RPMI
o For 50 mL stocks= 18 mL FCS + 27 mL RPMI
o Aliquots can be stored at -20°C
o Once thawed, store at 4°C and use within one week
Freezing medium 2 (20% DMSO + 80% FCS [heat-inactivated])
o For 15 mL stocks= 10 mL FCS + 2.5 mL DMSO
o For 50 mL stocks= 36 mL FCS + 9 mL DMSO
o Aliquots can be stored at -20°C
o Once thawed, store at 4°C and use within one week
10m
Isolation of PBMCs (via LeucoSept)
1h 50m
Fill up to 20 mL of whole blood (e.g. from two heparin tubes) into a 50 mL LeucoSep tube
(equipped with a divider and 15 mL of Ficoll).
Rinse the emptied blood tubes with 5-10 mL of PBS and add this to the LeucoSep tube.
Finally, fill the LeucoSep tube with PBS to approximately 40-45 mL.
All above procedures should be performed at room temperature.
Work quickly, as the separation medium (Ficoll) is cytotoxic.
Pictures and description from Greiner Bio-One Instruction manual LeucoSep
20m
The layers are separated by centrifugation at 1000 x g, 10 min, 20°C, without brake
Note
It takes about 35-40 min for the centrifuge to swing out.
This is a good time to prepare a cryo container (Mr. Frosty), the cell counting slides and maybe label your final cryo tubes.
45m
After centrifugation, the mononuclear cells (primarily lymphocytes and monocytes) are located in the interphase, which is recognizable as a whitish ring. Discard the plasma fraction above (stopp approx. 20mm above the whitish ring to not disturb the interphase).
Then, transfer the interphase from the LeucoSep tube into a fresh 50 mL Falcon tube by pouring. The divider disc (in the LeucoSep tube) prevents contamination with the depleted erythrocytes, granulocytes and Ficoll.
LeucoSep layers before and after centrifugation. Figure © ZeBanC; created with BioRender
Note
When transferring to a new Falcon, it is possible to combine two LeucoSep tubes (same blood draw; do NOT mix patients/timepoints).
15m
To remove Ficoll residues, the collected cells are washed with PBS by centrifugation.
For this purpose, the 50 mL Falcon tube (containing the interphase) is filled with 20-30 mL of PBS (@RT) (total volume approximately 40-45 mL), mixed by inverting several times, and then centrifuged at 400 x g for 10 min, medium break (@RT).
20m
After centrifugation, a cell pellet should be visible at the bottom wall of the Falcon tube.
Discard the PBS (pour off/remove by pipetting) without disturbing the cell pellet.
Note
After this step you can pre-cool the centrifuge to 4°C
5m
Resuspend the pellet in 10 mL of PBS (@4°C).
Mix the cells thoroughly with the PBS and immediately remove 10 µL for cell counting.
Note
While counting cells, you can already centrifuge the Faclon tube/s. See step 4
5m
Automated cell counting (via Countess3)
7m
For cell counting, 10 µl of the cell suspension is taken and mixed with 10 µl of trypan blue solution [ratio 1:1] (in a separate container/small tube).
Dead cells will be stained blue by trypan blue. 10 µl of the trypan blue-stained suspension is pipetted into an opening of the counting chamber and, after approximately 30 seconds, transferred to the automatic cell counter.
Steps for cell counting on the Countess3. © invitrogen/ThermoFisher
5m
The counting chamber is inserted into the slot of the device (Countess3 FL) with the loaded side (A or B) facing forward. The Countess 3 cell counter focuses automatically (our settings exclude everything under 8µM as cell depris or RBC). Counting begins after the Count button is pressed. The relevant result, including the number of viable cells, is displayed as Live (% and concentration per mL).
Inserting counting chambers into the Countess3 FL and starting the counting process. Pictures © ZeBanC
PS: The 1:1 dilution (step 3.1) with trypan blue is automatically factored in by the cell counter
Note
Approximately (0.5 – 3) x 10^6 PBMCs can be expected per mL of human whole blood.
For example, using two full heparin tubes (2 x 9 mL), we expect a total cell count of (2-4) x 10^7 cells [= 20–40 x 10^6].
Since we diluted to 10 mL of PBS (step 2.6), the cell counter should show between (2–4) x 10^6/mL. Ideally, over 90% of cells should be viable.
1m
Calculate the required volume of complete freezing medium to adjust to desired cell concentration.
Complete freezing medium consists of 50% freezing medium 1 and 50% freezing medium 2, which are added in a two-step procedure (see steps 4.1 and 4.2
Note
Example: A concentration of 2.99 x 10^6/mL is displayed and we previously dissolved the cell pellet in 10mL PBS (step 2.5), we have 10mL x 2.99x 10^6/mL = 2.99x 10^7 (i.e. 29.9 x 10^6) cells.
If we need a normalized concentration of 1x 10^6/mL (i.e. a million cells per mL), we would have to dilute the cell pellet (after the next centrifugation) with 29mL of complete freezing medium (=14.5mL freezing medium 1 + 14.5mL freezing medium 2).
Pictures + Figures © ZeBanC
1m
Freezing of PBMCs (using Mr. Frosty)
47m
Centrifuge the 50mL Falcon tube containing the cell suspension (in PBS, 10mL; step 2.5) at 400g, 10min, 4°C, with medium brake.
Note
Can be started during the cell counting.
10m
Discard the supernatant (PBS) & pipette off the return flow without disturbing the pellet.
Quickly resuspend the pellet with (50% total volume) of freezing medium 1 (consisting of 40% FCS + 60% RPMI, @4°C).
The pellet can initially be dissolved in a smaller volume (with a smaller pippette); however, the total volume remains the same.
3m
Then, dropwise, add (50% total volume) of freezing medium 2 (consisting of 80% FCS + 20% DMSO, @4°C) and mix within the Falcon tube by (slight) swirling motion.
If necessary, allow it to run along the inside wall of the Falcon.
Note
Since freezing medium 2 contains DMSO, you will have to work quick as soon as its added to the cells.
3m
Finally, transfer 1.9mL cell solution (each) into 2mL cryotubes. This leaves some room for expansion of fluids while freezing.
Note
At ZeBanC we like to use 2D-barcoded tubes like LVL, FluidX or similar.
1m
Take a cryo container (Mr. Frosty), transfer the filled cryotubes into the Mr. Frosty (@4°C) and then place the Mr. Frosty in a -80°C freezer. Note the time and date of freezing onto the lid.
The cells should be transferred to a liquid nitrogen tank (-160 to -196°C) within one week, but earliest after 24 hours of storage at -80°C.
Note
Prepare/check the isopropanol level in Mr. Frosty beforehand.
Some cryo containers do not need isopropanol.
10m
Finally, the data (tube ID, date of preparation, concentration...etc) is entered into the Biobank LIMS system
20m
Protocol references
Isolation:
https://www.gbo.com/fileadmin/imported_from_old/Leucosep_Protocol.pdf (see: Attachments); adapted
Time to processing:
Lehle S, Völkl S, Seitz K, Goossens C, Emons J, Ruebner M, Uhrig S, Ziegler P, Theuser AK, Beckmann MW, Fasching PA, Huebner H. Effect of delayed isolation of peripheral blood mononuclear cells on cell viability and functionality. BMC Immunol. 2025 Mar 15;26(1):21. doi: 10.1186/s12865-025-00701-y. PMID: 40089714; PMCID: PMC11909936.
Rivas I, Li Z, Irvin J, Huda N, Arzi B, Vapniarsky N. Delayed isolation and cryopreservation have significant effects on the yield and proliferation of feline peripheral blood mononuclear cells. Am J Vet Res. 2025 Jul 14;86(10):ajvr.25.04.0124. doi: 10.2460/ajvr.25.04.0124. PMID: 40675187.
Diane Longo, Brent Louie, Erik Evensen, Rachael E. Hawtin, Alessandra Cesano; Impact of Time From Blood Draw to Peripheral Blood Mononuclear Cell (PBMC) Processing and Cryopreservation on Functional Pathway Activity As Measured by Single Cell Network Profiling (SCNP) Assays. Blood 2011; 118 (21): 4922. doi: https://doi.org/10.1182/blood.V118.21.4922.4922
Browne DJ, Miller CM, Doolan DL. Technical pitfalls when collecting, cryopreserving, thawing, and stimulating human T-cells. Front Immunol. 2024 May 15;15:1382192. doi: 10.3389/fimmu.2024.1382192. PMID: 38812513; PMCID: PMC11133553.
Amelia Jerram, Thomas V. Guy, Lucinda Beutler, Bavani Gunasegaran, Ronald Sluyter, Barbara Fazekas de St Groth, Helen M. McGuire; Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples. Biosci Rep 26 February 2021; 41 (2): BSR20203827. doi: https://doi.org/10.1042/BSR20203827
Freezing media:
Juhl M, Christensen JP, Pedersen AE, Kastrup J, Ekblond A. Cryopreservation of peripheral blood mononuclear cells for use in proliferation assays: First step towards potency assays. J Immunol Methods. 2021 Jan;488:112897. doi: 10.1016/j.jim.2020.112897. Epub 2020 Oct 10. PMID: 33049298.
Storage Temp:
Yang J, Diaz N, Adelsberger J, Zhou X, Stevens R, Rupert A, Metcalf JA, Baseler M, Barbon C, Imamichi T, Lempicki R, Cosentino LM. The effects of storage temperature on PBMC gene expression. BMC Immunol. 2016 Mar 15;17:6. doi: 10.1186/s12865-016-0144-1. PMID: 26979060; PMCID: PMC4791795.
Central Biobank Charité would like to acknowledge the BIH (Berlin Institute of Health) CRU/BeLOVE Unit for sharing their SOP.
Acknowledgements
Central Biobank Charité would like to acknowledge the BIH (Berlin Institute of Health) CRU/BeLOVE Unit for sharing their SOP.