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PBMC's Isolation


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#1 kushekhar

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Posted 21 April 2009 - 09:05 PM

Hi There,
I wanted to know Which anti-coagulant and its working concentration used to separate PBMC and Plasma. I need to use PBMC's for culture and cytokine responce. Plasma to measure cytokine profile using CBA (cytometric Bead Array) as well as reverse Transcriptase PCR from the RNA extracted from the plasma. please can anyone suggest me the anti-coagulant and its concentration to used for separation of plasma and cells.

Kushekhar

#2 bob1

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Posted 22 April 2009 - 05:08 PM

You options are heparin or EDTA, I don't do this sort of stuff so I can't tell you how much to add.

#3 kushekhar

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Posted 22 April 2009 - 08:30 PM

You options are heparin or EDTA, I don't do this sort of stuff so I can't tell you how much to add.

Thank you bob 1, but I heard EDTA inhibit PCR? someone recomonded Sodium Citrate, but its concentration i dont know.
waiting for suggestions.

#4 Doki

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Posted 22 April 2009 - 11:32 PM

We use 0.1-0.2ml of Heparin for collecting 5-10ml of blood for immunocytometry or other procedures.

Not sure about EDTA and PCR but should not be a problem with Heparin I guess. U can run a control just to be sure. What procedure will you be using to isolate PBMC?
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#5 bob1

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Posted 23 April 2009 - 04:58 PM

A quick read suggests that heparin will co-precipitate with DNA and interfere with PCR as well. DNA extraction from EDTA coated tubes should be OK, most of the EDTA will be washed away in the wash step. Anyway, plenty of people re-suspend DNA in TE buffer and it works fine in PCR.

#6 Doki

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Posted 23 April 2009 - 07:05 PM

A quick read suggests that heparin will co-precipitate with DNA and interfere with PCR as well. DNA extraction from EDTA coated tubes should be OK, most of the EDTA will be washed away in the wash step. Anyway, plenty of people re-suspend DNA in TE buffer and it works fine in PCR.


This is new for me. I am sorry that I mentioned Heparin here though I had mentioned I use it for immunocytometry studies. I googled for Heparin-PCR and found a few studies. Picked up these for reference.

- Effects of heparin on polymerase chain reaction for blood white cells.
- EDTA Is a Better Anticoagulant than Heparin or Citrate for Delayed Blood Processing for Plasma DNA Analysis
- Direct PCR from Whole Blood

It would still be better to run a control to be on safe side whatever U might use.

Thx Bob1
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