sushmamanral wrote:
>> 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
> _______________________________________________
> Immuno mailing list
>Immuno from net.bio.net>http://www.bio.net/biomail/listinfo/immuno>>thank you I'll surely implement that and get back to you. ashis
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