Working on a protocol evaluating neo-antigen induced T-cell response with human PBMCs and plan to use and IFN-gamma ELISA.
I have two questions.
1) What peptides have people used as a positive control (in addition to a mitogen positive control like PHA CD3/CD28). Based on info provided by the company these PBMCs have 1-2 SFC from a CEF and CMV peptide pool. Tetanus? companies?
2) Has anyone used AIM V media (contains some human serum albumin) for human PBMC culture without further serum supplementation (Human or bovine)? Would be looking to stimulate with neo-epitope (controls) perform ELISA on day 5, and restim on d7 with additional IL-2 or IL-7 and perform 2nd IFN gamma ELISA on day 10-12
Thanks
1) Hi, related with positive control for human IFN gamma ELISA; Can you get the QuantiFERON®-TB Gold ?? this test uses a peptide cocktail simulating the proteins ESAT-6, CFP-10, and TB7.7. I've tried it several time with good results. Any way, you have to take in mind the population coverage of this antigens
.http://www.quantiferon.com/irm/content/PI/QFT/2PK/UK.pdf
2) for human PBMC culture, we've always supplemented with FBS 10%.
Thanks Frank! We had discussed using something related to Tetanus but wasn't sure what was available, will look into it!
For your PBMC culture, did you use AIM V or X-Vivo media? Had been hoping to use that as I thought it would decrease variables that might change the characteristics of my T-cells. ALso the other reason I had hoped to avoid adding FBS, but you've had good results with this? I did find one paper from 1988 looking at macrophage culture that stated you could use AIM v media for up to two weeks.
Thanks again for your response and any additional advice you can add
for PBMC culture, I use to RPMI 10% FBS, and recently we´ve tried with AIM V with 10% FBS. I´ve never tried without 10% FBS. we always obtain good results with our "home made" INF gamma ELISA.
I quest that isolation PBMC step is veer important for final results.
here the protocol that we use:
Isolation of Human T Lymphocytes
Obtain human blood from a healthy donor. Allow the blood to cool to room temperature (~30 min) before proceeding to the next step.
Gently pipette 3 mL of room temperature Polymorph density gradient media into an 8 mL round-bottom polystyrene tube. Gently add 3 mL of whole blood on top of the Polymorph media. It is important to avoid mixing of the two reagents.
Centrifuge the tubes at 500 x g for 45 minutes at room temperature.
Following the centrifugation, the peripheral blood mononuclear cells (PBMC) have now separated from other blood components into the top cell layer. The PBMC layer appears, from the top down, as the first cloudy band.
Carefully remove the clear yellow-colored upper phase of the blood, above the PBMC layer, and then use a P1000 micropipette to transfer the PBMC layer to a 15 mL or 50 mL conical tube.
Wash the PBMC twice with PBS, centrifuging cells at 500 x g for 5 minutes each time. The supernatant will be somewhat cloudy after each wash.
try to avoid the contamination using 1% penicillin/streptomycin.
you can scale down this without problems
good luck
Thanks again Frank! very helpful. We are actually purchased PBMC with specific haplotypes for some neo-epitopes we had synthesized.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA