The experiment was realized in 20 goats, the samples were taken every week, since october to december 2015. The sample were taken with heparinized vacutainer tubes and storage at 10ºC. I have never worked with flow cytometer. We want to stimate some parameters of the humoral response in this animals, I was thinking in cell populations but I dont know if the sample is still viable for this studies or some interleukin like IL-4, IL-6, IL-10. Thanks!!!
Similar with the other question, I think most of the samples are compromised as many of these epitopes are perishable. December blood may be perhaps usable for plasma although cells have probably died and passively released mediators.
Cells can be prepared for FACS by incubating with antibodies soon after drawing the blood, then the fixed cells with antibodies bound on it can be stored and FACS-ed at a later date.
I am totally agree with Mihai Oltean, the majority of the samples you stored there is a lot of time ago, then you might have probably lost all the markers. You are probable going to find IL-4, IL-6... but not to know which cell produce it, since your majority of cells would be dead, and losing the CDs.
Next time you want to make FACs with different pool of cells, the best idea is to make the staining in fresh when you obtain the samples, and then fix them. Once they are fixed, you can stored them for a while a 4`C and in the dark.
After depleting cells, you can test certain cytokines by ELISA or multiplexed bead array. The value would be lower than the fresh one, but still detectable. In my hand several freeze thaw samples did work.
Being a vet and working on ruminant immune system - sorry mate, no chance. Cells are dead after that time, so the only thing you can do is trying to analyse some cytokines by ELISA in plasma. You may struggle with that as well as not many ELISA for bovine cytokines are tested for any other ruminant species, and the same goes for antibodies to goat CD markers (in case you want to repeat). After 2h on ice, cells are starting to change surface antigen expression.
And there are hardly any commercially multiplex bead assays in veterinary immunology - we cant buy things of shelves, like the mousologists ;-)
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