[Immunology] NEED HELP

ashissr via immuno%40net.bio.net (by ashis.rasaily from gmail.com)
Thu Dec 25 00:54:05 EST 2008




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
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> 
thank you I'll surely implement that and get back to you.      ashis

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