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
