My answer is , Ive experienced during an serology lab the Rbc cells were lysed is pretty hard , ysing EDTA provide clots prevention , when while doing an (GIEMSA STAINING) thin smear in a slide with blood smear isn't well proper after add an 70% ethanol doesn't happen after I add the excess of the ethanol in placed on thick blood smear area , I found agglutination with lysis ,after I've observed under the microscope, they blood cells were damaged means RBC cells could irregular in shape in 40x resolution, So this is an one type method in lysis of RBC cells easily !

You can use another lysing solution like Turk's solution to lysis the RBC.

Hi Lisha van Onselen , after lysis your solution will still be red but it should be clear (transparent). You will notice the difference if you hold your sample up with some writing behind it. Pre-lyse, you should not be able to see through your sample, post-lyse you should be able to. You can then centrifuge your sample, take off the supernatant, resuspend your pellet, wash the cells, then resuspend in a buffer of your choice and perform flow cytometry.

We lyse cells at room temperature in the dark for 10 minutes using: https://www.thermofisher.com/order/catalog/product/00-4300-54

Our protocol for analysis of monocyte subsets on a flow cytometer:

Add antibody mixture to 100 uL whole NaH blood and leave in the dark for 10 minutes. We stain in 1.5 ml eppendorfs.

Fill all eppendorfs with lyse and leave in the dark for 10 minutes.

Centrifuge at 300 x g at 4 celsius for 6 minutes.

Resuspend the pellet and fill each eppendorf with fridge temperature FACS.

Centrifuge at 300 x g at 4 celsius for 6 minutes.

Resuspend in 400 uL PBS and perform flow cytometry.

Hope this helps.