for cell count in fluorospot assay

[Guest Seong Yeon]

I had asked several questions to Christian by e-mail. 

But I still have some questions in Christian's reply. Thus, I ask some questions again. 

 

Regarding positive control, you recommend 50,000-100,000 cells/well.

However, in your mabtech manual, recommended cell count is 250,000/well

Does it mean that cell count can be lower in positive controls than other wells?

If that is right, how appropriate cell count in negative control?

 

In the report of Weinberg (attached file),  they performed VZV specific dual fluorospot assay (IFNr/IL2)

In their report, cryopreserved PBMC were thawed and rested overnight (in your manual, at least 1 h is recommended)

Is there any differences? 

 

 

jiy383.pdf

[Christian@mabtech.com]

Dear Seong Yeon, welcome to the forum!

Yes, the cell count in positive control wells should in most cases be lower compared to the antigen specific wells. In fact, this is often how we do it at Mabech in our quality control for human IFNg, we run:

Unstim= 250,000 cells/well

CEF= 250,000 cells/well

PPD= 250,000 cells/well

PHA= 50,000 cells/well

AntiCD3= 50,000 cells/well

The positive controls have inherently higher spot counts at lower cell numbers simply due to the stimuli being "polyclonal" in its activation. By contrast, antigen specific wells only induce T-cells that are very rare in frequency, so the cell number needs to be higher. Since you are most interested in the antigen specific wells, the unstimulated control should match the cell count to that of the antigen specific wells, not the positive controls. 

In general it is smart if you initiate your study by doing a small "test" of like 3 donors. Do a titration of cells with your antigen. Test if it is better to incubate 24h or 48h. Include or exclude anti-CD28. The cell numbers above are for human IFNg. By contrast, for a cytokine like IL-17A, it is recommended to use 100,000 cells well in the positive controls as these T-cells are naturally lower in frequency compared to IFNg secreting T-cells. 

Yes, resting overnight will most often result in differences compared to only 1h. Some people say that you in this way, by resting overnight, “reset” the T-cells into a "tissue specific state" and that this is beneficial for antigen specific responses. I dont have great experience in doing the resting overnight so I cannot comment much. 

At Mabtech in our QC controls we do the 1h rest mainly due to getting rid of dead/apoptotic cells that would otherwise affect living cells within the culture during the assay. By removing the dead cells the "environment" is improved and you get better T-cell responses. 

 

[Guest Seong Yeon]

Thank you for your kind and reliable reply !!