NEED HELP

[ashissr]

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
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[sushmamanral]

On Dec 19, 11:01 am, ashis.rasa...@... 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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Immuno mailing list
Immuno@...
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[ashissr]

sushmamanral wrote:

On Dec 19, 11:01 am, [e-mail] 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
[e-mail]
http://www.bio.net/biomail/listinfo/immuno

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

[ashissr]

hi,
     could you tell me a good univ to do phd in immunology??i would be
very grateful.thank you                ashis
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