I am planing on using splenocytes and mesentric lymph nodes for cytokine elisa, does anyone have a protocl, that shows how many cells concentration for mice.
Hi,
There are several methods to isolate cells from both of these organs. An easy one is to obtain the organs, and using the plunger end of the syringe, mash them against a metal mesh or 100 um filter into a conical tube. The splenocytes will need a Red Blood Cell Lysis step. Rinse syringe plunger and filter and resuspend cells in 10 mL of media. Take 10 ul of cell suspension and add to 90 ul of cell membrane dye (we typically use Trypan ble or Eosin red). Take 10 ul of this cell/dye mixutre and plate onto a hemocytometer. Count cells through a microscope; at this dilution each cell counted will represent 1x10^6 cells. This will give you a pretty accurate count.
If you need a more detailed organ prep protocol I can provide one. Depending on what cytokine you are looking at / in what context the ELISA will vary. We typically employ ELISPOT as a technique for cell cytokine analysis in varying stimulation contexts.
Hope this helps!