fluorospot volume

[Guest sohyun lim]

Hi Mabtech!

I used your human IFN/IL2 fluorospot kit.

I ask about capacity of stimulator, because when using a ELISPOT kit from another company, they recommend 150ul in a well. 100ul cell, 44ul media, 6ul stimulator.

(stimulator is what I'm dealing with to see the reaction difference in same cells.)

I also use same volume in your kit, but when ask about it, I received another answer. total volume is 200ul, 100ul cell, 50ul media, 50ul stimulator.

My confusion is when I used as a previous volume in your kit, positive wells (I deal with stimulators in three ways, negative-control-positive) had a lot of reactions. To what extent, the greatest number of positive wells have over 1,000 spot in each wells, that I thought I did not need more volume of stimulator. Is not that a lot?

If i need to use more capacity than originally used, how can I do? use more stimulators? or dilute before use? 

 

Thank you for your help.

[Christian@mabtech.com]

Dear Sohyun!

The final volume in the ELISpot or FluoroSpot well does not really matter. However, you need to pick one and keep that consistent throughout your study. 

I recommend 200ul as the final volume since this is easier to calculate:

With 200 ul as the final volume you add cell stimuli at double the final concentration

Cells are added at 100ul/well. 

Everything becomes easier and the likelihood of mistakes goes down.  

Having 1000 spots per well in a postive control well is not uncommon. Maybe I am misunderstanding your last question. 

 

[Guest Seong Yeon]

Hello, Christian !

We performed Mabtech Fluorospot in each wells with 100 ul cell, 44 ul media, and 6 ul simulator. However, there was a problem with the results of the experiment. Although I think the main problem is that we didn't use anti CD28 mAb, however, we are also looking for the another possibility leading to wrong result. She had seen more than 1,000 spots in the positive spot even in a small 6 ul stimuli (PHA), and she seemed to be worried if it was raised to 50 ul, it would be too reactive. In case of VZV lysate, we previously used 6 ul, it would be too reactive. So,  it seems to ask if we need to dilute VZV lysate. 

I wonder if you recommend 200 ul because the capacity of the media and the stimuli is just good to calculate.

Do you have any other special reasons, such as the optimal concentration or mix of media? 

We had used MRC5 cell (mock infected), VZV infected MRC5 cell (VZV lysate, antigen), PHA (Positive control) once asked.

For negative control, you recommend the use of PBMC + media + anti CD28 mAb. Doing like this, I wonder if I could rule out the stimulation of MRC 5 cells. What is your opinion?  And how many spots are reasonable or acceptable for negative control well?

Thanks, 

[Christian@mabtech.com]

In my experience there are very few stimuli that can be added at too high concentration. With PHA for example, it does not matter if we use it at 2,5 ug/ml or 5ug/ml. Going up to 10ug/ml is no problem either. However, going down too low is not good and can result in no stimulation. Maybe the VZV lysate is different but are you sure? Have you attempted a dilution of this stimuli and actually confirmed that it stops working at higher concentrations?

When you say that you used 6ul if PHA stimuli, what as the final concentration in the ELISpot wells? This is a crucial aspect naturally. It is also strongly recommended that you first dilute the PHA in cell culture medium and then add this solution to the ELISpot/FluoroSpot wells. To me it sounds like you have added 6ul stimuli straight down into the wells? Easy to make mistakes pipetting such small amounts into a 96 well plate. 

200ul is my recommendation simply because it is easier: Easy to prepare the stimuli and their concentrations and also easy to calculate the number of cells added to each well. 

The anti-CD28 can cause some background in PBMC for some cytokines. Some more than others. Especially IL-2 tend to give a few spots background in the presence of anti-CD28. By contrast, IFNg backgrounds most often stay the same (i.e anti-CD28 does not increase IFNg secretion in unstimulated controls ).