preparing peritoneal macrophage - problem in lysing erytrocyte (Jun/21/2006 )

hi everyone

I have a problem in erytrocyte lsying method in peritoneal exudate by using hypotonic saline solution. Can you advice me about the appropriate time and temp. to use. My experiment has been prepared for macrophage function producing nitric oxide

blue

hi,

Give Chilled Distilled Water tretement.

Ashu

We use an ammonium chloride based method for removing red blood cells just incubate in it at RT for 5 mins. The recipe is already on the bioforum pages just search for "ammonium chloride" and you'll fine a few recipes for red cell lysis buffer.

Hi parkpoom,
We are also using hypotonic lysis of RBCs in peritonial macrophage.
for this we are using 10XPBS filters through .22u syringe filters take 100ul of 10XPBS in one pipettes and 900ul water in other and now add 900ul water in the cells just cont 1-10 sec and add
100ul of 10XPBS kept cells for 5 min at 4 temprature and add incomplete media and now tranfers its into other tube by avoining come up debries in this tube spin its at 300g for 5min agains repeat this protocole till you not able to get white pellets of the cells.
for further quiary you can ask me without any hesitation on my mail id awadh507@gmail.com