Anyone familiar with ELISA of cytokines in mouse plasma? - (Jan/19/2012 )
I am new to ELISA and I am going to measure TNF-alpha in mouse plasma (from heparinized blood).
The blood was collected from terminal bleeding and I got about 300~400ul plasma. I am not sure about what diluent I should use for diluting the samples.
Also, does anyone know about the basal level of TNF-alpha (in pg/ml) in mouse plasma when doing ELISA?
One option if you are unsure of the concentration is to start out plating samples twice - one at the neat concentration, one at 1 in 10 dilution (10ul + 90ul) - using the same standard. If the concentration is particularly high the neat plate will develop very quickly, but the 1/10 dilution should remain within the standard curve. If you're really unsure you can do further dilutions 1/100, 1/1000 etc. Do this on a small scale so as to not waste your sample.
For diluting you can use the same solution you use to dilute the standards (commonly called Assay Diluent in kits) or normal cell media (RPMI) have both worked fine for me.
When it comes to analysis I've got a plate calculator available here that allows you to put different plate dilutions in.