Researchers collect human peripheral blood mononuclear cells (PBMCs) from blood samples for use in various research 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.

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. Ficoll™ or Lymphoprep™) and centrifugation. This method takes advantage of the differences in density between the cells in blood and the density gradient medium.

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 Lymphoprep™.

Advantages to using density gradient centrifugation to isolate PBMCs include the low cost of the density gradient media, lack of specialized equipment, 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.

Find more efficient ways to process blood samples >

Faster Density Gradient Centrifugation Using SepMate™ PBMC Isolation Tubes

SepMate™ is an innovative tube that enables consistent and hassle-free PBMC isolation in a 15-minute protocol. SepMate™ simplifies the density gradient centrifugation protocol, resulting in fewer errors and reduced variability between users. You can quickly layer blood over the density gradient medium because the SepMate™ 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.

Isolation of Highly Purified PBMCs Using Magnetic Cell Separation

Skip centrifugation and isolate PBMCs directly from blood in as little as 20 minutes using EasySep™ Direct Human PBMC Isolation Kit. This kit immunomagnetically depletes red blood cells (RBCs), platelets, and unwanted cells in a single step without density gradient centrifugation. Untouched PBMCs are simply poured into a new tube and are immediately available for downstream applications such as flow cytometry or cell culture.

How does EasySep™ 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 1).

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 EasySep™ Direct Human PBMC Isolation Kit results in reduced granulocyte contamination compared to density gradient centrifugation (see Figure 1).

3. Enables automated PBMC isolation
EasySep™ Direct Human PBMC Isolation can be automated using the RoboSep™-S 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 and increase sample throughput.

Learn more about RoboSep™ cell separation instruments >

Try the EasySep Direct Human PBMC Isolation Kit in your own lab.

Sourcing Fresh or Frozen Human PBMCs

Laboratories looking for a reliable supply of mononuclear cells can source human PBMCs from primary cell suppliers. Large lots of cryopreserved PBMCs with specific donor criteria (e.g. sex, age range, BMI range, ethnicity, CMV status, HLA type, diseases, etc.) can be secured, thereby ensuring consistency across multiple experiments.

Alternatively, researchers can obtain blood products such as leukopaks and Leukocyte Reduction System (LRS) cones, which contain large numbers of mononuclear cells collected from human peripheral blood. Learn how to process leukopaks and LRS cones for the isolation of mononuclear cells:

• Protocol: Processing a Leukopak for Downstream Cell Isolation
• Video: How to Recover Cells from a Leukocyte Reduction System (LRS) Cone

View all human PBMC products >

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 at liquid nitrogen temperatures (below -135°C) until needed. When the cells are needed, the cryopreserved PBMCs can be thawed and then used in downstream applications.

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: