Hi.
I am about to fine-tune the ELISPOT kit to quantify the number of antigen-specific ASCs. But to start I need to know if:
- For blocking and culturing cells, can RPMI medium with phenol red be used? Does the red color of this medium interfere with the development of the test?
- In what volume should the cells be added to the well?
Thank you so much! greetings
Hello!
You can absolutely use RPMI 1640 with phenolred in ELISpot. No issue.
I recommend you always add stimuli first in ELISpot wells (100ul) and then afterwards add in cells (100ul) for a total volume of 200ul/well during the incubation. It is ofcourse also possible to do 50ul+50ul for total volume of 100ul, but I generelly find this option more sensitive to evaporation effects and it increases the likelihood of making a calculation effect. It is easier for the brain to calculate the 1/10 dilution effect when pipetting 100ul, compared to the 1/20th calculation needed for 50ul....