PBMC isolation from buffy coat samples
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