Hello, Hassan Muhammad Rostam Al-Bewani

I have to share with you a document that is very suitable to answer your question.

https://www.nature.com/articles/s41598-022-24550-6

The key information in the literature is as follows:

Core markers for distinguishing monocytes, macrophages and neutrophils in human blood samples

1. Monocytes Classical markers:

CD14 (main marker), CD16 (subpopulation distinction), HLADR (co-expression). Subpopulation classification: Classical monocytes: CD14hi CD16neg/lo; Inflammatory monocytes: CD14hi CD16+; Non-classical monocytes: CD14lo/mid CD16+ (Document Figure 1). Auxiliary markers: HLADR+ (distinguished from neutrophils), CD66b- (excluding granulocytes).

2. Neutrophils Core markers:

CD66b+, CD16++, CD14neg/lo, HLADR- (Document Figure 1). Physical characteristics: high forward scatter (FSChi) and high side scatter (SSChi), but scattered light changes may occur after transportation due to activation/degranulation (Document Figure 2).

3. Macrophages Macrophages in the blood:

There are fewer macrophages, and they are usually differentiated from monocytes. The markers can refer to CD68+ and CD163+, but they need to be combined with the tissue source (the document does not mention it directly, and additional supplements are required). Note: Macrophages in the blood are often classified as monocyte subsets. If specific distinction is required, additional antibodies (such as CD68) are required.

Antibody strategies for different cells

• Recommended antibody combinations: FITC-anti-CD16, PE-anti-CD14, PerCP-Cy5.5-anti-CD3, APC-anti-HLADR, AlexaFluor700-anti-CD66b.
• Principle: It can exclude T cells (CD3+), mark monocytes (CD14, CD16, HLADR) and neutrophils (CD66b, CD16).

Gating strategies for different cells

Step 1: Singlets

Based on FSC-A (area) vs. FSC-H (height), exclude cell adhesions.

Step 2: Exclude lymphocytes and T cells

• Lymphocytes: FSClow SSClow, further confirmed and excluded by CD3+.

Step 3: Distinguish granulocytes from monocytes

• Granulocyte gate: FSChi SSChi, further confirm neutrophils with CD66b+ HLADR-.
• Monocyte gate: FSCmid SSCmid, circled with HLADR+ CD66b-, and then subdivided by CD14/CD16.

Step 4: Monocyte subset subdivision

Among HLADR+ CD66b-CD14+ cells, with CD16 as the horizontal axis and CD14 as the vertical axis:

• Classical monocytes: CD14hi CD16neg/lo;
• Non-classical monocytes: CD14lo/mid CD16+;
• Inflammatory monocytes: CD14hi CD16+.

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