unfortunately I can not use centrifugation. 

Can you recommend a good lysis recipe?

Maybe Becton Dickinson - BD Pharmlyse™ Lysing Buffer could be an alternative for you. It works fine without any centrifugation step involved, lysing your RBCs and letting your PBMCs intact.

add your sample of blood to ACK (at least 20 volumes for full RBC lisys), mix and wait 5-7 min at room temperature to solution became clear (transparent).

If this diluted cell suspension is good for your future investigation.

Don't keep cells long time in ACK - they will start apoptosis or NETosis. And keep in mind what amonium cation influences phagocytosis.

http://escore.im.wustl.edu/SubMenu_protocols/protocol8.html

Add water will do it.  It is cheap and it works everytime.

Sergio

If you use water don't forget to recover isotonic conditions after

Thank you all for your good advice.

I will try and get back to you with the results.

See previous discussions:

https://www.researchgate.net/post/RBC_lysis_buffer_for_Flow_Cytometry_without_Fixation

https://www.researchgate.net/post/Can_anyone_recommend_a_RBC_lysis_recipe_for_mouse_blood

https://www.researchgate.net/post/Do_you_know_why_my_RBC_lysis_solution_is_not_working

https://www.researchgate.net/post/Can_anyone_recommend_a_good_RBC_lysis_protocol_for_isolation_of_PBMCs_from_rat_blood

Use ACK lysis buffer.

Add milliQ just for 30 seconds or 1 minute and then add 1X PBS to it and spin down , you will get a pale white pellet of PBMCs.

I usually use water and it work perfectly for me

For chicken embryo fibroblast cell culture, how to remove RBC from sample. I want to know the procedure using lysis buffer.

Shamsudeen Lekan Pedro Sergio Torloni

Hello ;

Can you say the protocol for using water? please tell me about amount of water and centrifuge speed