Whole blood sample
Separation of distinct layers
Isolation of PBMCs
PBMC isolation from buffy coat samples
- Isolate the buffy coat from whole blood samples, or procure them from a biorepository.
- Read our protocol for buffy coat isolation from whole blood here
- If you need help counting your cells, we have outlined a protocol in the next section of this article
In this protocol3, we will describe how you can manually estimate the number of PBMCs in your sample using trypan blue and a hemocytometer or similar chamber.
1. Prepare a 1:1 dilution of your cells with 0.4% trypan blue in a 2 mL tube.
- Pipette the solution up and down to mix
2. Incubate cells in this mixture for 2-3 minutes.
- Prepare the hemocytometer while the cells are incubating with the mixture
3. Transfer 10 µL into the counting chamber and count the number of alive (non-blue) cells and dead cells (blue) using a light microscope.
- Use this number to estimate the total number of viable cells in the original sample
- Bittersohl et al. Intracellular concentrations of immunosuppressants. Personalized Immunosuppression in Transplantation (2016).
- Cottler-Fox et al. Collection and processing of marrow and blood hematopoietic stem cells. Hematopoietic Stem Cell Transplantation in Clinical Practice (2009).
- Bidmon et al. Tumor Immunology and Immunotherapy - Cellular Methods Part A. Methods in Ezymology (2020).
Editors note: This protocol is for guidance only, based on freely available information and typical protocols used in labs worldwide. REPROCELL is not responsible for the results of any work using this protocol(s).