i am preparing buffer for RBC lysis,
using following chemicals
NH4cl : 8.0 gm
NaCo3 : 1.0 gm
EDTA : 0.1 gm
in 100 ml water
Sir, i want know the role of each one of these chemicals?
will i get clear or turbid solution?
I want to remove RBCs from whole blood, and interested in lymphocyets.
It is better to write down the molarity of the solution. Probabily it is an iperosmolar solution. EDTA is a chelating agent to remove Ca++ from the blood and avoid coagulation if blood is too concentrate in the solution. The Ph of your solution should be around 9. You will get a clear solution if the RBC are broken. To isolate lymphocytes use commercial kit, you'll need a centrifuge.
Thank you Mr Roberto..
Yes i am treating it with RBC lysis buffer and then centrifuge so as to remove RBC cell debris from it (by aspiring the supernatent)
just yesterday i have prepared this solution and its pH is around 8. i hope it work for RBC lysis and getting good number of WBCs for culturing with RPMI 1640 medium ....what you think will these WBCs servive after treatment of such Buffer and centrifugation?
Hi We use this RBC Lysing solution (10x) - In 1L dH2O NH4Cl 80.2 g, NaHCO3 8.4 g, EDTA disodium 3.7g, dilute 1:10 in dH2O to make working solution. To use add 1X RBC lysis buffer, incubate 10 minutes al RT, making sure to mix the pellet well by pipetting, after 10 minutes spin for 10 minutes 1200 rpm, 4°C to remove RBC lysis buffer
Thank you for your nice replies..For what purpose you do lyse RBCs...? for karyotyping or else..?
Do you mean Isolation..?
Will WBCs survive after this RBC lysis buffer treatment for further culturing and growing them..?
oh, yes isolation
Yes, PBMCs are fine, but this treatment is for just 10 minutes no longer.
We isolate monocytes from leukopak, if you need the full protocol, let me know.
I was successful in lysisng RBCs in last week, today i am going to culture the cell mass( i.e. WBCs) left after rbc lysis...
do anybody have suggestion regarding that...
I am using RPMI 1640 for culturing blood cells..
hii Sham ashok Patil....can u please tell me if i can use this rbc lysis buffer for dilute blood sample
@mukta, RBC lysis buffer is used when someone wants to reduce or remove RBC from blood sample, may I know on what exactly you are working?
Dilution of blood sample can be done in many ways depending upon the ultimate aim of your experiment.
Sham...actually i already had dilute blood samples...and i have to extract DNA from....during isopropanol precipitation i m getting rbc debri....so i want to get rid of rbc before DNA extraction.....so i m trying to use RBC lysis buffer....will it work....please reply as soon as possible
Yes it works, its also available in market, I used self prepared, but you can purchase it from Hi-Media/ sigma.
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