Isolation of Peripheral Blood Mononuclear Cells from Human Buffy Coats

Abstract

Source: Mily, A., et al. Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection. J. Vis. Exp. (2020)

In this video, we demonstrate the isolation of peripheral blood mononuclear cells, PBMCs, containing monocytes and lymphocytes from a human buffy coat by density gradient centrifugation. The isolated PBMCs can be used for further analysis.

Protocol

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 Mt....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lymphoprep	Alere Technologies AS	11508545
RPMI 1640	Life Technologies Corporation	SH30096.01
EDTA (0.5 M)	Karolinska University hospital, Huddinge	N/A
Na-pyruvate	GE Healthcare Life Sciences	SH300239.01
L-Glutamine	GE Healthcare Life Sciences	SH30034.01
HEPES	GE Healthcare Life Sciences	SH30237.01
Fetal bovine serum (FBS)	Sigma-Aldrich	F7524