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Flow cytometry and cytokines


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4 replies to this topic

#1 NCIS is science fiction

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Posted 14 January 2011 - 08:02 AM

Hello all I am new to the forum as well as the procedure I am attempting. (Moderators feel free to move me if I am in the wrong place :D )

I have been looking for a precise directions on how to complete my task, but I have found bits and pieces never the whole picture. I am attempting study human inflammatory cytokine levels with a CBA kit, but collecting samples for processing is a big question to me. I have the following questions: Keep in mind these are all leading up to using a Cytometric Bead Array Kit.

1. Should we use serum or plasma? ( From readings I am leaning towards serum)
2. How much should we obtain from subjects for use? (The kit says we should be able to use 50~100 ul to obtain results, but how much should we collect from the subjects?)
3. How should we store the serum or plasma after collection? (What types of tubes should be used for collection and storage? and why?)
4. Can we store plasma or serum for extended periods without a heavy effect on the results? ( We would prefer collecting many samples then running them through flow cytometry)
5. After all of these starting point the procedure seems straight forward from the CBA kit instructions, do you have time/sample saving tips for using these CBA kits?

Thanks all.

#2 DRT

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Posted 16 January 2011 - 08:32 PM

Are you intending to stimulate the blood ex vivo? Is this a clinical trail or are you looking for in vitro effectors?

With clinical trials; we collect 4mL heparin blood. Immediately(ish) dilute 1:1 with RPMI then stimulate for a couple of hours before spinning out and storing the plasma at -80deg.

With in vitro experiments (usually only one donor) we use a ficol prep to extract the PBMC cells prior to stimulation and assay the samples with CBA on the same day.


#3 NCIS is science fiction

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Posted 18 January 2011 - 08:08 AM

We will only be looking at cytokine serum levels. It is a clinical study (not trial)but we plan on collecting a certain number of samples (multiple donors) before we actually use the CBA kits. That is why I wanted to know what would be the best method of collection and storage.

Thanks.

#4 Denis Baev

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Posted 19 January 2011 - 03:19 AM

I have been looking for a precise directions on how to complete my task, but I have found bits and pieces never the whole picture. I am attempting study human inflammatory cytokine levels with a CBA kit, but collecting samples for processing is a big question to me. I have the following questions: Keep in mind these are all leading up to using a Cytometric Bead Array Kit.

1. Should we use serum or plasma? ( From readings I am leaning towards serum)
2. How much should we obtain from subjects for use? (The kit says we should be able to use 50~100 ul to obtain results, but how much should we collect from the subjects?)
3. How should we store the serum or plasma after collection? (What types of tubes should be used for collection and storage? and why?)
4. Can we store plasma or serum for extended periods without a heavy effect on the results? ( We would prefer collecting many samples then running them through flow cytometry)
5. After all of these starting point the procedure seems straight forward from the CBA kit instructions, do you have time/sample saving tips for using these CBA kits?
.

1. Doesn't matter.
2. Depends of your estimations about the cytokine concentration. The CBA has a minimal and maximal ranges for detection so that sometimes you need to dilute your analyte before the CBA staining procedures to be done.
3. Frozen in LN or even at -20 are ok
4. Yep, in LN
5. Follow the Kit Manual and DONT FORGET TO AJUST A COMPENSATION FIRST

Good luck!

#5 NFkb

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Posted 02 March 2011 - 09:42 PM

Hi,
Carefully follow kit manual,
you would be alright.


But my serum is very limited (mouse serum ) less than 50 ul. I have seen someone using CBA kit with 25 ul of PE, test sample and bead mixture for all the results.

Anyone can suggest me how shall i proceed with very small serum samples. It was recommended that Assay diluent can be used for dilution, but the manufacturer emailed me telling that if we dilute, they cannot gurantee the results.

Plus, I found 2 kit (post date expiry) unused in my department and stored at 4 degree Celsius.
Can those be used as well ??

Thanks
Milan




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