Isolation of Peripheral Blood Mononuclear Cells from Human Buffy Coats

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Preparation of media

NOTE: Details about all the reagents and consumables are provided in the Table of Materials.

1. RPMI complete medium: Supplement RPMI 1640 with 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, and 10% heat-inactivated fetal bovine serum (FBS). Avoid antibiotics in the cell culture medium when working with Mtb infection.
2. Serum-free RPMI medium: Supplement RPMI 1640 with 1 mM sodium pyruvate, 2 mM L-glutamine, and 10 mM HEPES.

2. Peripheral blood mononuclear cell isolation from buffy coats

NOTE: Perform all work with human blood (potentially contagious) inside a class II biosafety cabinet. Inactivate residual blood products with disinfectants for 15 min before discarding. Blood was obtained from healthy volunteers in this case. This in vitro macrophage differentiation protocol was set up to include 10 x 10⁶ isolated PBMCs/donor/well. From each donor, one buffy coat contains about 50 mL of a concentrated leukocyte suspension originating from whole blood, which normally provides 500–800 x 10⁶ PBMCs from which approximately 10% or 50–80 x 10⁶ monocytes can be retrieved.

1. Load 15 mL of buffy-coat blood on top of 15 mL of density gradient medium prepared in a 50 mL tube. Slowly overlay blood on top of the density gradient layer by leaning the pipette tip to the wall of the tube.
2. Spin the tubes at 600 x g for 25 min at room temp (RT) with 0 acceleration and 0 deceleration.
   NOTE: Close lids carefully before centrifugation and always check the tube holders for potential spillover after centrifugation.
3. Remove the top plasma layer with a sterile Pasteur pipette and thereafter carefully collect the mononuclear cell layer into a new 50 mL tube using a sterile Pasteur pipette. Add serum-free RPMI medium to the PBMC pellet to obtain a final volume of 50 mL. Mix carefully by inverting the tube a few times before centrifugation at 500 x g for 5 min at RT.
4. Discard the supernatant carefully and resuspend the cell pellet by flipping the bottom of the tube within the fingers.
5. To remove the density gradient medium contamination from the PBMCs, wash cells 2–3 times with serum-free RPMI to obtain a final volume of 50 mL. Centrifuge at 500 x g for 5 min at RT. Wash until the cell supernatant becomes transparent.
6. Discard the supernatant and resuspend the cells in 20 mL of serum-free RPMI medium.
7. Count the cells by trypan blue staining, manually using a hemocytometer, or using an automated cell counter. Dilute the cell suspension in trypan blue in 1:2 or 1:10 dilution by mixing the cell-trypan blue sample in a 96-well plate e.g., 50 µL + 50 µL (for hemocytometer counting) or 10 µL + 10 µL (for automated cell counting) and count the cells to obtain the number of live cells/mL.
   CAUTION: Trypan blue is toxic and must be discarded in a separate chemical waste.