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Cell Biology JoVE (Open Access) Citable · DOI

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

DOI: 10.3791/57941-v
What you'll learn
  • ✓ Prepare whole blood samples for flow cytometry analysis
  • ✓ Gate monocyte subsets using CD14 and CD16 markers
  • ✓ Quantify M1/M2 inflammatory marker expression on monocytes
  • ✓ Validate gating strategy and interpret monocyte phenotyping results
Protocol

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Difficulty
intermediate
Total time
~2–3 hours per sample (including sample preparation, staining, and acquisition)
Model organism
Human (whole blood)
Biosafety
BSL-2

Steps

1
Prepare whole blood samples for flow cytometry

Process fresh whole blood samples and prepare them for staining. This includes handling and preprocessing steps to ensure cell viability and proper preparation for antibody labeling.

▶ 00:42
2
Perform flow cytometry staining and acquisition

Stain prepared blood samples with fluorescently labeled antibodies targeting monocyte markers and run samples on a flow cytometer. Optimize instrument settings and collect data.

▶ 02:14
3
Gate monocyte subsets by flow cytometry

Apply sequential gating strategy using CD14 and CD16 surface markers to identify and isolate classical, intermediate, and non-classical monocyte subsets from whole blood.

▶ 03:28
4
Validate gating and quantify marker expression

Assess accuracy of monocyte subset gating and measure expression of M1 (inflammatory) and M2 (anti-inflammatory) markers on each subset. Analyze and interpret results.

▶ 07:11
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