Jun 10, 2026
703 and 704. Whole Blood Peripheral Blood Mononuclear Cell (PBMC) Isolation, Freezing and Thawing. LungMAP HTC SOP
- Ravi Misra1,
- Gautam Bandyopadhyay2,
- Heidie Huyck1,
- Sally Quataert1,
- Gloria Pryhuber1
- 1University of Rochester Medical Center;
- 2Department of Pediatrics, University of Rochester Medical Center
- Ravi Misra: Lung MAP HTC - BioRepository for Investigation of Neonatal Diseases of the Lung (BRINDL);
- Heidie Huyck: Lung MAP HTC - BioRepository for Investigation of Neonatal Diseases of the Lung (BRINDL)
- Sally Quataert: URMC Immunology
- Gloria Pryhuber: Lung MAP HTC - BioRepository for Investigation of Neonatal Diseases of the Lung (BRINDL)
- Human BioMolecular Atlas Program (HuBMAP) Method Development Community
- LungMap2 Consortium
Protocol Citation: Ravi Misra, Gautam Bandyopadhyay, Heidie Huyck, Sally Quataert, Gloria Pryhuber 2026. 703 and 704. Whole Blood Peripheral Blood Mononuclear Cell (PBMC) Isolation, Freezing and Thawing. LungMAP HTC SOP. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr1e6pgmk/v1
Manuscript citation:
Misra RS, Bhattacharya S, Huyck HL, Wang JC, Slaunwhite CG, Slaunwhite SL, et al. Flow-based sorting of neonatal lymphocyte populations for transcriptomics analysis. J Immunol Methods. 2016;437:13-20.
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: June 06, 2026
Last Modified: June 10, 2026
Protocol Integer ID: 318634
Keywords: peripheral blood mononuclear cells, PBMC, whole blood, plasma, serum, human peripheral blood mononuclear cell, peripheral blood mononuclear cell, whole blood peripheral blood mononuclear cell, retrieval of peripheral blood mononuclear cell, flow cytometry those rbc, diluted blood on the ficoll, cell types such as pbmc, pbmc for sample, red blood cell, blood from young infant, immature red blood cell, diluted blood, premature infant blood volume, anticoagulant, pbmc, platelet, collected blood, contaminating rbc, flow cytometry, yield of pbmc, successful cell recovery from the frozen state, cell sample, laboratory assay, blood, higher density granulocyte, rbc lysis step, successful cell recovery
Funders Acknowledgements:
National Institute of Heart Lung and Blood
Grant ID: U01HL148861
NIH Common Fund
Grant ID: U54HL165443
Disclaimer
NOTE: If reagents listed on Page 1 are unavailable, then the use of an equivalent reagent is acceptable.
Abstract
Purpose and Scope of the Procedure or Laboratory Assay
- Scope: To outline the retrieval of peripheral blood mononuclear cells (PBMC), from whole blood collected in anticoagulant, using Ficoll-Hypaque density gradient centrifugation. This protocol was used to isolate PBMC for samples received for LungMAP HTC and HuBMAP MOSDAP Project.
- Scientific Principles:
- Human Peripheral Blood Mononuclear cells (PBMCs) and platelets have a lower density than Ficoll-Hypaque (1.077 g/L) and are separated from higher density granulocytes and red blood cells when centrifuged after either under or overlaying the diluted blood on the Ficoll-Hypaque layer. Blood from young infants may contain immature red blood cells that may contaminate the upper buffy coat layer after centrifugation. The contaminating RBC may be removed from the infant buffy coat by a second Ficoll-Hypaque centrifugation step. However, since premature infant blood volumes are so small, optimizing the yield of PBMCs/ml of collected blood is critical. So a second ficoll step is not recommended. An RBC lysis step and an anti-RBC surface marker (CD252) will be used to remove and identify in flow cytometry those RBC not removed from the cell sample.
- To retain cellular integrity, we recommend that cells be frozen at a slow controlled rate of minus one degree Celsius per minute in cryoprotectant medium containing 10% dimethylsulfoxide (DMSO). Use of 90% FBS improves post-thaw viability in our experience. The DMSO acts to prevent ice crystal formation during the slow freezing process, maintaining cell viability. Some cell types such as PBMC may be stored short term (less than one week) at -800C or long term in a liquid nitrogen freezer.
- For maximum recovery and viability upon thawing, cells should be healthy prior to freezing. Successful cell recovery from the frozen state requires rapid but gentle thawing of the cells followed by immediate dilution and removal of cryoprotectant medium from the cells. Note the protocol suggesting thawing only 1-2 vials at a time and initial slow, 1-2 drop, addition of buffer to avoid shocking the cells.
Materials
- Hanks’ balanced salt solution without phenol red, calcium or magnesium (HBSS)(Cellgro Catalog #21-022-CM) or equivalent
- Fetal Bovine Serum (FBS)(screened for low cell cytotoxicity and of quality to support cell growth with low endotoxin)
- DMSO, Dimethylsulfoxide (reagent grade) Sigma Cat # 15,493-8 or equivalent
- Ficoll-Paque‱ Plus (Amersham Pharmacia Biotech Catalog # 17-1440-03) or Lymphocyte Separation Medium (Cellgro Cat# 25-072-CV) density of 1.077 g/liter at 20°C or equivalent
- Dulbecco’s PBS, Hyclone, Cat#SH30028.03 or equivalent
- - ACK Lysing Solution, BioWhittaker Cat#10-548E or equivalent
- 0.04% Trypan blue exclusion dye in saline
- Household bleach (5% sodium hypochlorite), 10% bleach solution
- Equipment/Model:
- Biological Safety Cabinet, Class II
- Beckman Allegra X-14 centrifuge with SX4750A swinging bucket rotor and inserts with BioSafety Caps or equivalent
- Sterile serological pipets 50, 25,10, 5, 2, 1 mL size
- Pipet aide for serological pipets
- Sterile pipet tips
- Single channel air displacement pipets 5-50 mL
- Sterile 15mL or 50mL conical polypropylene tube
- Test tube racks for 15mL or 50mL conical tubes
- -Cryovial, Nalgene Nunc/5000-1020
- Cryogenic vial racks
- Cell counting tube
- 80°C freezer and liquid nitrogen freezer for long-term storage
- Waste pan
- Absorbent towels
- Sterile containers for dilution of blood (ie conical tubes)
- Ice and bucket
- Mr. Frosty, Nalgene Cryo 1°C Freezing container Cat# 5100-0001
Safety warnings
Personnel will adhere to safe work processes outlined in U.S. Public Health Universal Precautions Guidelines for use of human blood and body fluids. PPE will be used including lab coat, closed shoes and gloves. All activity will be behind shield of biosafety cabinet and/or with mask and safety glasses. Biosafety level 2 practices will be followed and the work performed in the designated lab space which is covered by annually updated IBC approved protocol. All institutional biosafety measures are followed in any manipulation of these human tissues.
Before start
Note: All work is performed using BSL 2 procedures and following universal precautions for handling human blood and body fluids, in designated lab space, approved by annual review of IBC.
Samples should be processed within 24 hours of being drawn, in most cases, as soon as possible after sample arrives at HTC lab
Prepare Reagents
Cryprotectant/ Freezing medium (10% DMSO / 90% FBS (eg. Gibco Cat#10437-028)): Sterile filter FBS using a 0.2 micron filter with vacuum into a sterile container to remove any particulate matter. Add 10 ml DMSO per 90 ml FBS using a sterile disposable pipet. Be careful not to introduce the pipet beyond the tip to ensure ink on the serological pipet does not enter the DMSO. Do not re-filter after addition of the DMSO. Store the medium at 40 C. Always use sterile technique when entering the solution container. Place the cryoprotectant medium container on ice while using to freeze cells.
Freezing container: A Mr Frosty with isopropyl alcohol to the fill line is used to control the rate of cell freezing in the -800C freezer. Mr Frosty containers are cleaned and new alcohol added after every 5 uses. Mr Frosty is stored at 40 C prior to freezing cells to ensure that cells do not warm as they are placed into the holder prior to placing in the -800 C freezer.
For Thawing: Select One:
*** FACS media = 1% BSA in DPBS, w/v (GB)
Or, if cells going into cell culture, suggest to use Enzyme Neutralization buffer = 10% FBS in DPBS (RM) or DMEM + 10% FBS.
Serum recovery and storage
Serum is recovered from whole blood samples collected in a red top or similar tube that does not contain an anticoagulant. The blood is allowed to clot at room temperature without dilution, centrifuged and serum aliquots removed and frozen in cryovials at -800C.
Process blood to isolate plasma and PBMCs using Ficoll Hypaque
Dilute whole blood with 2 parts of 1X Dulbecco’s phosphate buffered saline to 1 part whole blood in a sterile container. For example, for 5 ml of blood, add 10 ml of DPBS
Layer 10 mL of diluted blood over 3 mL of Ficoll-Hypaque in a sterile 15 mL conical.
If there is less than 10mL of diluted blood for layering, use a similar ratio of diluted blood to ficoll as stated previously. For this process, it is critical to use the volumes indicated above to prevent cell loss. Layer over the Ficoll-Hypaque by dribbling the diluted blood slowly down the side of the tube using a pipet,to prevent mixing of the diluted blood with the Ficoll.
Place tubes in centrifuge cups, that have been equilibrated to 20°C, in balanced positions and attach aerosol containment lids. Centrifuge at 1200X g for 30 minutes at 20°C with no brake
After centrifuge has stopped, place sealed cups carefully within a BSL2 hood. Remove tubes being careful not to disturb the white cell layer containing the mononuclear cells on top of the ficoll layer. Cells are kept at 4˚C following harvesting to maximize cell viability.
Freeze plasma samples: Remove plasma top layer and add 1 mL to each of 3+ cryovials. Enter sample into Inventory and print Freezerbond labels. Attach 1 label/vial. Place at -80°C and record in the freezer management log.
Then, remove the white blood cells layer by siphoning with a 2 or 5 mL pipet. Place the cells in a sterile 15 ml polypropylene tube.
Fill tube containing cells with refrigerated HBSS to the 14mL line and centrifuge the harvested cells at 300 X g (1100 RPM) for 10 minutes at 4˚C. Pour off supernatant into waste container.
Tap up cells in the pellet and wash 3 additional times by resuspending cells in 14mL of refrigerated HBSS and centrifuging at 300 X g (1100 RPM) for 10 minutes at 4˚C to remove platelets. Cells are kept at 4˚C following harvesting in order to maximize cell viability.
Resuspend PBMC in volume of HBSS to provide cell density appropriate for counting.
Dilute 10µL of cells in 40µL ACK lysing solution and incubate at RT for 5 minutes.
Add 50µL 10% Trypan Blue solution. Count viable (trypan blue negative) and non-viable cells on hemacytometer (or alternate method) using 1/10 as the dilution factor. Record cell count. Expect between 0.5x10^^6 and 7x10^^6 PBMC/mL of blood.
Calculations:
% viability = live / live+dead X 100.
Cells/mL = 10 X (live/# squares counted) X 10^^4^^
Aliquot and freeze recovered cells
Follow the table below to determine the number of cells to be frozen per vial.
Total Cell number / number of cells to be frozen per vial =# mL of freezing media.
Round cells to the nearest vial.
Example: (21.0*10^^6) cells equals 4 vials at 5.2(10^^6^^/vial) then resuspend in 4 mls freezing media
| A | B | C | |
| Total Number of Cells Recovered (x106) | # vials to freeze | Concentration of Cells per Vial (x106) | |
| <8 | 1 | All Available | |
| 8-10 | 2 | Split equally between vials | |
| >10-50 | 2-10 | 5x106 cells per vial | |
| >50 | 10 | Xx106 cells per vial divide cells evenly into 10 vials |
Spin the cells at 300 X g for 10 minutes at 4°C and pour off the HBSS.
After centrifugation, pour off supernatant and resuspend cell pellet by gently tapping the tube by hand (with fingers) before adding the appropriate amount of freezing medium to tube to yield a cell concentration/vial as indicated in table. Immediately pipet 1 ml of cells in cryprotectant medium into labeled cryovials, close caps tightly and place into a chilled Mr.Frosty. Transfer the Mr. Frosty to a -800 C freezer.
After 24 to 72 hours, transfer the frozen cell aliquots from the -80°C freezer to liquid nitrogen freezer tank. Record in the freezer management system, the date and location of the samples placed in LN2 storage.
Procedure for thawing cells
Move tubes quickly to dry ice from liquid nitrogen freezer for transporting to laboratory.
Thaw only 1-2 tubes at a time in 37o C water bath while shaking gently by hand continuously just until the last ice crystal remains. This should take just over one minute for vials containing 1 ml of freezing medium.
Using a one ml pipet, transfer the entire contents from the tube to a 15ml conical polypropylene tube at ambient room temperature. Slowly add 5ml of cold FACS media or enzyme neutralization buffer, 1-2 drops at a time, while mixing carefully and thoroughly by swirling tube for the first 5 ml. Repeat for the second vial (if applicable).
Add an additional 5 ml of warm media to each tube before proceeding directly to next step.
Centrifuge tubes at 1000 X g for 10 min at 40C. Remove supernatant, gently tap tube to resuspend cell pellet and add 10 ml warm FACS media or enzyme neutralization buffer. Centrifuge the tube again as above. Discard supernatant.
Resuspend the cell pellet to desired volume in FACS or culture medium. Dilute an aliquot of cell suspension in trypan blue exclusion dye for counting in a hemocytometer (or alternative method) to determine recovery and viability.
Use resuspended cells subsequently in single cell assay or culture.
Protocol references
Current protocols in Immunology (2009) 7.1.1-7.1.3 John Wiley & Sons, Inc.
Recommendations for Prevention of HIV Transmission in Health-Care Settings: Universal Blood and Body Fluid Precautions Guideline CDC 1987
CDC Biosafety in Microbiological and Biomedical Laboratories 4th edition U.S.H&HS, Public Heath
Service
HIC-1-0007.2:Cryopreservation of cells: Freezing and thawing
HIC-3-0023: Clinical Specimen Management Policy
HIC-3-0025: Clinical Specimen Receipt, Storage, Testing, Processing,Distribution, Archiving and Disposal.
HIC-1-0034.2 and 604-PROP-1-090111