[Immunology] Re: NEED HELP

sushmamanral from gmail.com via immuno%40net.bio.net (by sushmamanral from gmail.com)
Tue Dec 23 10:45:35 EST 2008


On Dec 19, 11:01 am, ashis.rasa... from gmail.com wrote:
> THANK YOU FOR YOUR SUGESSTION LIT LI CHEING.I NOW HAVE A PROBLEM REGARDING  
> THE FICOL PROCESS.ie FOR MY PROJECT IMMUNOMODULATORY EFFECTS OF ALOR VERA  
> EXTRACT ON HUMAN PBMCS,I GET THE BLOOD[ WITH EDTA] AND AFTER 15 MINUTES I  
> USE IT FOR FICOL GRADIENT SEDIMENTATION. I DILUTE THE BLOOD WITH EQUAL VOL  
> OF PBS BUFFER TO AVOID QUICK CLUMPING WHICH USED TO EARLIER HAPPEN IN THE  
> NEXT STEP ie WHEN I ADD FICOL.
> I USUALLY HAVE, 2, 14 ml TUBES OF THIS FICOL-BLOOD SO THE TIME TAKEN FOR ME  
> TO CENTRIFUGE AFTER I HAVE FILLED THE BOTTLE IS APPROX 5-6 MINUTES. I  
> CENTRIFUGE IT IN A SWINGING BUCKET CENTRIFUGE FOR ABOUT 20-25 MINUTES AT A  
> SPEED OF ABOUT 3000 rpm. I THEN GET MY BUFFY COAT.
> I NOW SEPARATE MY BUFFY COAT USING A PIPETTE.i .e I FIRST REMOVE THE  
> SERUM/PLASMA ON THE VERY TOP FOLLOWED BY THE BUFFY COAT.I WOULD NOW LIKE TO  
> SPECIFY THAT ALONG WITH THE BUFFY COAT I REMOVE EVEN SOME PARTS OF FICOL  
> UNDERNEATH IT AND SOME PARTS OF THE SERUM ABOVE ONLY TO OPTIMISE THE AMOUNT  
> OF THE BUFFY COAT.I THEN ADD EQUAL VOL OF PBS BUFFER TO "WASH" THE BUFFY  
> COAT OR IN IN OTHER TERMS TO PRECIPITATE THE BUFFY COAT."NOW COMES THE  
> PROBLEM" I OFTEN GET A "RED SPEC" SOMETIMES BIG AND SOMETIMES SMALL, OVER  
> MY BUFFY COAT.NOW WHATS THAT?
> PS:AT THIS POINT I MUST SAY THAT MY CENTRIFUGE IS NOT THAT FINE ie IT HAS  
> ITS JERKS AS IT ROTATES BUT COULD THE "RBC" WHICH I KNOW IS AT THE TOP OF  
> YOUR MIND GO AGAINST THE GRADIENT AT A SPEED OF 3000 RPM? I AM REALLY  
> LOOKING FORWARD FOR YOUR ANSWER AND SHOULD I GO FORWARD WITH MY EXPERIMENT  
> OF SEEKING THE ACTION OF ALOE EXTRACTS ON THE "PBMCS" IF I GET THE RED  
> SPEC? AM REALLY LOOKING FORWARD FOR YOUR REPLY.WISHING YOU "ALL", THE BEST  
> IN YOUR WORKS ASHIS

hi
i m new to this site n happened to just read ur problem
actually i m also doing work on pbmcs or rather to say on lymphocytes
the red specs u get may be RBCs
u can give osmotic shock to remove this
add 0.2%NaCl to the pellet wait for 30 secs and then add an equal
volume of 0.16% NaCl dropwise to it and then centrifuge
give a wash after that so as to remove any NaCl.
hoping this may solve ur problem


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