Human Peripheral Blood Mononuclear Cells: Sourcing, Isolation, Freezing, and Thawing
Researchers collect human peripheral blood mononuclear cells (PBMCs) from blood samples for use in various applications, including flow cytometry, cell isolation, cell culture, and cell-based assays. Learn more about PBMCs and find protocols and tools to help you isolate, source, freeze, and thaw human PBMCs for your research.
PBMC Composition
PBMCs include lymphocytes (i.e. T cells, B cells, and NK cells), monocytes, and dendritic cells, and are defined as white blood cells with round nuclei. Preparation of a PBMC fraction from whole blood is a common step prior to the isolation of specific immune cell subsets.
Figure 1. Mean Percentages of Cell Subpopulations in Cryopreserved PBMCs
Representative chart showing the average frequencies of major immune subsets in cryopreserved PBMC products, as measured by flow cytometry post-thaw. Values shown are mean percentages of total viable leukocytes present in PBMCs (n ≥ 183).
Free Wallchart: Frequencies of Human Cell Types in Blood-Related Sources
Get a list of the estimated frequencies and percentages of more than 35 cell types in normal human blood-related sources.
Sourcing Fresh or Frozen Human PBMCs
If you’re looking for a reliable supply of mononuclear cells, º£½ÇÆƽâ°æ provides large lots of cryopreserved PBMCs, available in barcoded cryovials, with specific donor criteria (e.g. sex, age range, BMI range, ethnicity, CMV status, HLA type, disease state, etc.), thereby ensuring consistency across multiple experiments. Alternatively, choose º£½ÇÆƽâ°æ’s blood products, such as leukopaks and Leukocyte Reduction System (LRS) cones, which contain large numbers of mononuclear cells collected from human peripheral blood.
Diseased PBMCs are available from a large donor pool of patients with cancer, autoimmune, and inflammatory diseases, offered in convenient sizes for your research. To learn more about choosing fresh vs. frozen PBMCs and how º£½ÇÆƽâ°æ can support your specific PBMC needs, visit: www.stemcell.com/human-pbmcs
- Protocol: Processing a Leukopak for Downstream Cell Isolation
- Video: How to Recover Cells from a Leukocyte Reduction System (LRS) Cone
- Video: How to Process a Leukopak for Downstream Cell Isolation
Table 1. Available Cell Counts for PBMC Products
| PBMC Source | Available Cell Counts (Frozen) |
|---|---|
| Normal* | 5 million cells 15 million cells 25 million cells 100 million cells |
| Diseased: Cancer | 5 - 19 million cells |
| Diseased: Autoimmune and Inflammatory | 10 million cells 50 million cells |
*Fresh PBMCs are also available in 10 million and 30 million cell formats. Fresh products are available in select territories; to see our full fresh product offering and list of available diseased cell products, visit this page.
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Frequently Asked Questions on Primary Cells
Find answers to frequently asked questions on primary cells from º£½ÇÆƽâ°æ Technologies, including donor information, validation, and delivery options.
Learn More >Contact our primary cells team to learn more about how we can help you meet your research goals with fresh or frozen human primary cells.
Preparing and Isolating PBMCs from Whole Blood
There are several methods for collecting and preparing PBMCs from human whole blood, each with its own advantages and disadvantages. Learn about three methods below.
Basic PBMC Isolation Protocol Using Density Gradient Centrifugation
The most common PBMC isolation method involves using a density gradient medium (e.g. Ficollâ„¢ or ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢) and centrifugation. This method takes advantage of the differences in density between the cells in blood and the density gradient medium.
How to Isolate Mononuclear Cells from Whole Blood by Density Gradient Centrifugation
To isolate mononuclear cells from human peripheral blood, cord blood, and bone marrow, it is recommended to use a medium with a density of 1.077 g/mL, such as ³¢²â³¾±è³ó´Ç±è°ù±ð±èâ„¢.
Advantages to using density gradient centrifugation to isolate PBMCs include the low cost of density gradient media, minimal equipment requirements, and ease of introduction to any lab. However, this protocol is time-consuming, laborious, and cannot be automated. Isolating PBMCs from one sample can take at least 45 minutes.
Faster Density Gradient Centrifugation Using ³§±ð±è²Ñ²¹³Ù±ðâ„¢ PBMC Isolation Tubes
³§±ð±è²Ñ²¹³Ù±ðâ„¢ is an innovative tube that enables consistent and hassle-free PBMC isolation in a 15-minute protocol. By simplifying the density gradient centrifugation protocol, ³§±ð±è²Ñ²¹³Ù±ðâ„¢ reduces errors and variability between users. You can quickly layer blood over the density gradient medium because the ³§±ð±è²Ñ²¹³Ù±ðâ„¢ insert prevents the layers from mixing. Centrifugation is performed for only 10 minutes with the brake on, after which isolated PBMCs are simply poured off into a fresh tube and washed.
³§±ð±è²Ñ²¹³Ù±ðâ„¢: Hassle-Free PBMC Isolation Protocol
See how ³§±ð±è²Ñ²¹³Ù±ðâ„¢ works and request a sample to try it in your own lab.
Isolation of Highly Purified PBMCs Using Magnetic Cell Separation
PBMCs can also be easily isolated directly from blood without centrifugation or lysis using the EasySepâ„¢ Direct Human PBMC Isolation Kit. This simple and fast immunomagnetic isolation method results in purified PBMCs, in as little as 20 minutes, from whole blood, cord blood, bone marrow, buffy coats, leukapheresis samples, or a leukoreduction system (LRS) chamber (also known as an LRS cone). This kit immunomagnetically depletes red blood cells (RBCs), platelets, and unwanted cells in a single step without density gradient centrifugation. Untouched, highly purified PBMCs with reduced contamination from unwanted cells are simply poured into a new tube and are immediately available for downstream applications such as flow cytometry, cell culture, and more. Explore the benefits of EasySepâ„¢ Direct and see how it compares to traditional isolation methods.
EasySepâ„¢ Direct Human PBMC Isolation Kit
See how EasySepâ„¢ Direct magnetic cell separation works and learn more about how you can obtain highly purified PBMCs without lysis or centrifugation using EasySepâ„¢ Direct Human PBMC Isolation Kit.
To easily isolate PBMCs or different cell subsets manually from large-volume samples, including leukopaks and whole blood, choose the Easy 250 EasySep™ Magnet with EasySep™ reagents to scale up your isolations with ease. Instead of splitting your cell suspension and performing multiple rounds of isolation, you can process samples of up to 225 mL and 12.5 × 10⹠cells in as little as 20 minutes with a single isolation.
Automated Isolation of Highly Purified PBMCs with RoboSep™—Powered by EasySep™
Need to isolate a large number of highly purified PBMCs quickly and reproducibly but don’t have the time? Maximize efficiency and automate your PBMC isolations with RoboSepâ„¢. Use the column-free EasySepâ„¢ Direct Human PBMC Isolation Kit for RoboSepâ„¢ on the ¸é´Ç²ú´Ç³§±ð±èâ„¢-³§ to easily obtain highly purified PBMCs that are ready for downstream applications—all while reducing hands-on time, eliminating cross-contamination, and increasing sample processing throughput.
¸é´Ç²ú´Ç³§±ð±èâ„¢-³§: Automated Cell Isolation for Up to 4 Samples
See how the ¸é´Ç²ú´Ç³§±ð±èâ„¢-³§ fits easily into the workflow of any lab that needs the multi-sample processing capacity, speed, reliability, and convenience of automated cell isolation.
Try Automated PBMC Isolation in Your Own Lab!
See for yourself how RoboSepâ„¢ can enable you to isolate PBMCs efficiently and with minimal hands-on time.
How does EasySepâ„¢ Direct PBMC isolation compare to density gradient centrifugation?
1. Reduces platelet contamination
Density gradient centrifugation doesn’t remove platelets, which can easily become activated. Removing platelets requires additional time-consuming washing steps after the initial density gradient centrifugation. The EasySep™ Direct Human PBMC Isolation Kit removes platelets during cell isolation (see Figure 2).
2. Reduces granulocyte contamination
Granulocytes in blood samples degranulate and change density over time. Processing older blood samples (>48 hours after collection) using density gradient centrifugation may result in granulocyte contamination. By specifically targeting unwanted cells for removal with antibody complexes and magnetic particles, the EasySepâ„¢ Direct Human PBMC Isolation Kit results in reduced granulocyte contamination compared to density gradient centrifugation (see Figure 2).
3. Enables automated PBMC isolation
EasySepâ„¢ Direct Human PBMC Isolation can be automated using the ¸é´Ç²ú´Ç³§±ð±èâ„¢-³§ instrument automated cell separation instrument to minimize sample handling and free up valuable hands-on time. By automating all sample labeling and magnetic separation steps you can perform simultaneous cell isolations from up to 4 samples with as little as 5 minutes of required hands-on time.
Figure 2. EasySepâ„¢ Direct Human PBMC Isolation Kit Results in Fewer Contaminating Cells Compared to Density Gradient Centrifugation
Mononuclear cells were isolated from whole blood samples using either density gradient centrifugation or EasySep™ Direct Human PBMC Isolation Kit. Cells were counted and analyzed by flow cytometry. (A) Both density gradient centrifugation and EasySep™ Direct Human PBMC Isolation Kit resulted in an equivalent total number of nucleated cells recovered from 24-hour blood samples (mean ± SD; n = 14). (B) Using EasySep™ Direct Human PBMC Isolation Kit to obtain mononuclear cells from 24-hour-old blood samples resulted in lower numbers of residual platelets (CD41+), red blood cells (Glycophorin A+/CD45-), and granulocytes (CD66b+) compared to density gradient centrifugation (mean ± SD; n = 15). (C) Cell isolation from 24-hour, 48-hour, 72-hour, and 96-hour-old blood samples using EasySep™ Direct Human PBMC Isolation Kit resulted in lower numbers of residual granulocytes compared to cell isolation using density gradient centrifugation (mean ± SD; n=3).
Curious about how EasySepâ„¢ can simplify your research? Experience how easy the EasySepâ„¢ Direct Human PBMC Isolation Kit is to use by requesting a sample. Say goodbye to complicated protocols and time-consuming, column-based cell isolations. Make the easy choice and move your research forward faster.
Freezing and Thawing Human PBMCs
Researchers working with human cells can store frozen vials of isolated PBMCs for use in future assays (e.g. flow cytometry). To cryopreserve PBMCs, the cells are resuspended in cryopreservation medium, cooled to extremely low temperatures, then stored in the vapor phase of liquid nitrogen temperatures (below -135°C) until needed. When the cells need to be thawed for use, the ThawSTAR® CFT2 (Catalog #100-0650) offers a more standardized option to manual thawing. Although effective, manual thawing is also heavily operator-dependent, and small variations can significantly impact PBMC viability, function, and experimental reproducibility. ThawSTAR® CFT2 provides researchers with a more reproducible and GMP-compliant approach to PBMC thawing—ideal for applications in cell therapy, immune profiling, and multi-site studies where consistency is critical.
How to Cryopreserve PBMCs with CryoStor® CS10
Learn how to safely freeze human primary cells in ready-to-use, cGMP-manufactured CryoStor® CS10 for long-term storage.
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How to Thaw Frozen Primary Cells
Learn the proper technique for thawing frozen cells. Ensure optimal viability, recovery, and functionality of your cells for downstream applications.
View Protocol >Ensure high cell viability and functional stability following storage and thawing by using a reliable cryopreservation medium. Explore our cell storage media, including cGMP-manufactured and serum-free CryoStor® Freezing Media.
Downstream Applications: Using PBMCs in Your Research
Researchers use PBMCs for many downstream applications, including further isolation of specific cell subsets (e.g. T cells, B cells, monocytes), cell culture, and assays (e.g. flow cytometry).
Find products and technologies to support your specific downstream applications:
Antibodies
Cell Culture Media and Supplements
Cell Isolation Products
