The use of a lyse/fix solution prior to antibody staining is a simple and robust method. Data sheets from the companies have all the relevant information on them for you to follow.

Good luck!

Most of the protocols are pretty standard (both surface and intracellular). Almost any company that sells the antibodies will have a protocol for you to use.

The easiest is to lyse the RBCs and than stain.

The volume of whole blood need is usually only 100uL.

If you don't want to lyse the RBCs... well... the staining is still the same... but the analysis will require some one with more experience.

The staining protocol isn't the issue, the issue will be the analysis protocol on the flow machine. I would just ask someone who is well versed in flow to help you.

I generally use 100 ul blood/Heparin solution and directly add Abs.

Keep at 4C for 30 min and then wash 2X with 3 ml 1X Fix/Lyse solution (BD).

This is enough for surface staining.

As your objective is to look for leukocytes then all leukocytes express, CD45,

You can further differentiate cells by type T cells (CD3), B cells (CD19) DCs and Mac (CD11b), DCs (CD11c) and neutrophills (Ly6G).

Depending on your objective, you can add select or all antibodies, to the 100 ul Blood and folow stainig and lysis.

best

S

Dear Harpreet,

I used to try with Biolegend on Novocyte flow cytometer. results was so clean.

Please prepare your unstain and single stain for compensation purpose

You can try the brief protocol below :

- Add Abs to 100ul of whole blood (volume depends)

- Incubate in RT, 30min, dark

- Add 1X RBC Lysis buffer (volume depends)

- Incubate in RT, 15-20min, dark

- Sample is ready to proceed.

If you want to remove the debris, wash twice with FACS staining buffer, then suspend in 500ul-1ml buffer before analysis.

Best wishes to your experiment.