Issues with Elispot

[Saswati Emma Mohr lab]

I am doing experiment with Cryo saved PBMCs, with water soluble peptides from Miltenyi, and UV irradiated Virus. With exception to few samples almost most of the time the Elispot assay will not produce any spot with either the AntimCD3 or any of the stimulants with the saved PBMCs from the liq N2 or samples from minus 80 . Any ideas to see why this might be happening and suggestions for ways to fix? Attached here is the power point from my latest experiment with a total of 6 ID
Details in brief. Blocks of 4x3 wells, with respectively i)media,  ii) inactivated virus, iii) water soluble Peptides and iv)  anti CD-3 antibody  rows A, B, C D (1,2,3). Repeated the same scheme for the 2nd IDs  in respectively rows E, F, G H (1,2,3)
Move onto rows A, B, C D (4,5,6) and E, F, G H (4,5,6 ) for the 3rd and 4th ID. Move onto rows A, B, C D (7,8,9) and E, F, G H (7,8,9) for the 5th and 6th ID. Total 6 IDs
Other Information The difference between the 1st ID to the rest is that the first ID is collected fresh, and the other 5 are banked over 2- 3 years earlier. The number of cells used  for 1st ID is 2x10^5 cells
Also, letting you know,  I am not expecting spots from media / Virus/ peptides from the 1st ID , and as expected over multiple experiments the 1st Id responded to Anti CD3. So I consider it positive control to Anti-CD3
However  that none of the other IDs  respond to the Anti CD3 is where I want your feedback> The 2nd page of the power point has the lay out of the lane that I described in words in the first page. Please let me know if you have question on the lay out or anything in particular That I did not included here. Thanks a lot in advance. 
Saswati Emma Mohr lab


We had previous communication with you as of 
https://forum.mabtech.com/topic/871-problems-with-central-membrane-white-spot-and-retained-blue-color/

Presentation1 1st scan.pptx

[Christian@mabtech.com]

Dear Emma,

I have taken a look at your PPT and read your text. Slide 2 of the PPT and your text dont seem to add up in terms of the layout. 

According to the PPT slide2, in well D1, well D2 and well D3 there should be: rh2746 new

However, according to your text there should be antiCD3. What is correct? 

 

When it comes to crypreserved PBMC the viability is absolutly crucial for ELISpot to work well. Once you start getting below 81-84% viability you get suboptimal results. At 50-60% viability results can often be blank. 

Have you done any assessment of your PBMC when it comes to viability? Trypan blue exclusion for example. 

When explaining your experimental setup it is good if you work in different "layers". Please provide the layout of donors in one sheet. Then show cells/well in one sheet. Finally show stimuli in one sheet. 

I hereby share this experimental setup layout I like to use!

 

 

ELISpot Layout.xls