Elispot

[Guest So Young]

Dear Christian,

 

I am new to Elispot and trying to measure CD8 T cell reactivity using IFN-gamma and spleen cells from mice immunized with peptides.

1. From literatures search, I found that some scientists use peptides as stimuli and others co-incubate spleen cells with irradiated, peptide-coated syngeneic APCs as stimuli. Is it necessary to use APC pulsed with peptide to stimulate T cells? Or is it just to intensify the readout?

2. What is the Elispot plate without underdrain for?

3. What kind of stimulus would you use as a positive control? ConA, PMA, PHA?

 

Thank you for your advice.

 

So Young

[Christian@mabtech.com]

Welcome to the forum,

 

1. It is not necessary to use APCs pulsed with peptides to stimulate T-cells, but it may, like you say intensify the response (i.e get more spots). Notice that I say "may". This is not a black or white world. In addition, by introducing APCs you also introduce the variablity of how to "prepare them". Give them different mixes of stimulating agents and they can present the antigens in different cytokine contexts within the "immunological synaps", which will lead to differences in results. If I were you I would start by first trying your peptides without the APCs. If that does not work, move on to the APC pulsed approach. 

 

2. The ELISpot plate without underdrain, called MAIPSWU10 by Millipore, has been around for quite some time and was originally designed just for the purpose of creating a plate without an underdrain attached to the backside. You see, if the underdrain is attached to closely to the PVDF membrane you more easily get leakage following EtOH pretreatment. By designing the MAIPSWU10 without an underdrain, this "weak" point was essentially taken away.

 

The MAIPSWU10 is increadibly resilient when handled properly. You pretty much cannot get it to leak. However, since the plate sits in an removable undertray, it is possible to take it out, flip it around for washing and by mistake get small splashes of liquid on the backside of the membrane. If this happens, the MAIPSWU10 can also start leaking. 

 

As a result, the plate stays very resilient as long as you protect the backside of the membrane. I do this by keeping the undertray and plate "as one" during the assay by holding them together with the grip of my hand. This is talked about 5:30 into our EtOH activating video on youtube:

 

 

 

3. At Mabtech we use ConA with mouse cells. We like that the best.

[Guest Guest]

Thank you very much, Christian!