Hi,
I am staining a human blood with CD19, CD3,CD14 and CD16. My gating strategy in FlowJo goes like this:
- First, I gate a general population SSC-A VS FSC-A => Then gate for single cells, => after that I try to identify Lymphocytes, Monocytes, and Neutrophils.
- Then, I look for CD16 VS CD19 in Lymphocytes gate to identify the B cells, NK cells and T cells by gating first on CD14- then CD16 VS CD19.
- In the monocytes gate, I try to distinguish the monocytes sections( classic, non-classic and intermediate monocytes) by using CD16 VS CD14 markers.
- Lastly, in the Neutrophils, I gate on CD14 -- and CD19-- to identify CD16.
I am not sure about this gating strategy I found it a bit complicated and I am wondering if there is any sample way to identify the Lymphocytes, Monocytes, and Neutrophils in a human blood using this 4 markers!
Also, my problem is that I find it some time difficult to identify the monocytes and to distinguish the monocytes sections( classic, non-classic and intermediate monocytes).
Do you have any advice and tips to improve the staining and the gating strategy for better identifying the monocytes and Lymphocytes?
Thank you for your time and help.
Arwa
The CD16± NK cells is problematic. Cells often have small populations that change shape, eg neutrophils found in monocyte or lymphocte size area, meaning your CD16± gate may not be reliable for NK(CD56, is most ideal) To clear up the B cell and monocytes from the rest, good to add HLADR.
Regarding the monocyte subsets, this is usually influenced by time it takes to process blood and the anticoagulation type.
Dear Arwa,
Given colour limitations, your gating is very good.
The CD16± NK cells is problematic. Cells often have small populations that change shape, eg neutrophils found in monocyte or lymphocte size area, meaning your CD16± gate may not be reliable for NK(CD56, is most ideal) To clear up the B cell and monocytes from the rest, good to add HLADR.
Regarding the monocyte subsets, this is usually influenced by time it takes to process blood and the anticoagulation type.
The CD16± NK cells is problematic. Cells often have small populations that change shape, eg neutrophils found in monocyte or lymphocte size area, meaning your CD16± gate may not be reliable for NK(CD56, is most ideal) To clear up the B cell and monocytes from the rest, good to add HLADR.
Regarding the monocyte subsets, this is usually influenced by time it takes to process blood and the anticoagulation type.
Thanks Charlie Bridgewood for your answer.
I was thinking about changing the voltage of FSC VS SSA. Could this help in separating the small populations?
Also, can you please elaborate on how the time can influence the monocyte subsets or could you provide me with papers about this matter?
Thanks a lot for your help
Arwa
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