ReproCELL Inc.

08/09/2022 | News release | Distributed by Public on 08/09/2022 03:35

Protocol for PBMC isolation from buffy coat samples


PBMC isolation from buffy coat samples


1. Collect your buffy coats one day prior to the assay.
- Isolate the buffy coat from whole blood samples, or procure them from a biorepository.
2. Dilute the buffy coat with PBS 1:1 in a 50 mL conical tube and gently mix by inversion.
- Use a separate conical tube per donor
3. Dispense 15 mL of room temperature Histopaque®-1077 solution into a 50 mL conical tube.
- Pour 35 mL of the blood/PBS on top of the Histopaque®-1077 solution
- Avoid mixing them so that you have 2 separate phases
4. Centrifuge the tubes at 900xg for 22 minutes at room temperature.
5. Add 10 mL PBS into a 15 mL tube and transfer the PBMC layer from the centrifuged tubes without affecting the interphase or aspirating the Ficoll medium.
- Invert the tubes gently to mix
6. Centrifuge the 15 mL tubes containing the PBMCs-PBS mixture at 250xg for 5 minutes.
- Dispose of the supernatant
7. Resuspend the pellet in PBS and centrifuge at 250xg for 5 minutes at room temperature.
- Dispose of the supernatant
8. Repeat washing steps 6-7 if the supernatant is still cloudy (optional)
9. Resuspend the washed pellet at 2x106 cells/mL (if you need help counting your cells, we have outlined a protocol in the next section of this article) in a complete RPMI medium warmed to 37ºC.
- Store cells in a 5% CO2 incubator
- Add 50 μL interleukin-2 if needed (optional)

Protocol for counting PBMCs

In this protocol1,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 and 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 100 µL into the counting chamber and count the number of alive (non-blue) cells using a light microscope.
- Use this number to estimate the total number of cells in the original culture

References

  1. Bittersohl et al. Intracellular concentrations of immunosuppressants. Personalized Immunosuppression in Transplantation(2016).
  2. Cottler-Fox et al. Collection and processing of marrow and blood hematopoietic stem cells. Hematopoietic Stem Cell Transplantation in Clinical Practice (2009).

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).