Aug 06, 2019
PBMC Isolation from apheresis collars
Forked from PBMC Isolation
- Girija Goyal1,
- Girija Goyal2
- 1Wyss Institute for Biologically Inspired Engineering, Harvard University;
- 2Wyss Institute, Harvard University
Protocol Citation: Girija Goyal, Girija Goyal 2019. PBMC Isolation from apheresis collars. protocols.io https://dx.doi.org/10.17504/protocols.io.56bg9an
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 06, 2019
Last Modified: August 06, 2019
Protocol Integer ID: 26531
Abstract
Commonly used protocol to isolate peripheral blood mononuclear cells from whole human blood modified here for apheresis collars
Guidelines
Objective: Isolate peripheral blood mononuclear cells from platelet apheresis collars.
Safety warnings
Any materials that come into contact with blood should be sterilized with 10% bleach before discarding
Before start
- Make sure to repeatedly label sample with donor number, especially if working with multiple donors
- The protocol here is optimized for 10ml of material from platelet apheresis collars. Variations for other sources have been described.
Acquire collar and make an incision to drain blood into 50mL conical tube.
Dilute blood product with 2X volume RPMI or PBS. Mix well.
Slowly layer solution on top of 10 mL density gradient solution.
Centrifuge at 300 g for 25 minutes at room temperature. Set acceleration and deceleration levels to minimal.
22 °C
Remove white layer of PBMCs using a 5 mL pipette tip.
Add these cells to 10 mL warm media in a 50 mL tube.
If using 5 ml or more of the Leukopak, you may have a very high number of cells. To effectively wash them, fill tube to 50 mL.
Centrifuge at 120 g for 10 minutes to remove platelets and get an accurate count. Return acceleration / deceleration levels to high or 9.
Aspirate media and resuspend cells in 20 mL warm media per 10 ml of starting blood product. Steps 10-12 can be optimized depending on your yield.
Dilute 100x by adding 10ul cell solutions to 890 ul media in a 1ml eppendorf tube and 100ul Trypan blue
Count cells using a hemocytometer. Count the number of cells in each of the four quadrants. Use the following formula to find the total number of cells.total # of cells = (cells counted /4)X dilution factorX 104 cells/ml
Cells can be kept in solution in the refrigerator for up to two hours.