Hi all,
I have frozen (-20C) samples of whole blood from mice from a recent experiment. I need to find a way to thaw them so as to extract sera for an ELISA, while minimizing hemolysis and hopefully preserving serum proteins.
The samples are ~300uL whole mouse blood and stored in 1.5mL Eppendorf tubes without EDTA or heparin or anything.
I thawed one tube on ice and it was VERY clear that massive hemolysis had taken place (see picture of the supernatant collected after centrifugation; 10Krpm, 10min, 4°C).
I will be sitting corner with my dunce cap if anybody needs me.
Erythrocytes are fragile and will undergo hemolysis if frozen and thawed, as you demonstrated. If you need to freeze your samples to process them at a later time, you should let the blot clot in a glass tube, the collect and keep the supernatant (serum) frozen until needed. Now I don't know what you want to do with the blood, nor whether massive contamination with hemoglobin will impair your results. But it would be probably better to start the whole experiment once again (sorry for the mice).
Hi Jackson, well basically I agree with Pierre that the optimal way would have been the let the blood clot for about 1h at RT while freshly collected (as you have no anticoagulant), centrifuge to get the serum supernatant and freeze it. Now want you can do is to take fresh blood again from one mouse if available and redo the same as you did before. Then you let the blood thaw at 35°C and see whether you get a clot. If so, centrifuge the tube and collect the supernatant, which presumably will be reddish due to inevitable hemolysis since the whole blood was frozen (so the RBC will undergo lysis). You should get a supernatant what could be assimilated to a serum but contaminated by hemoglobin. I cannot judge whether your ELISA will be affected by the hemoglobin... You may know it by making the same experiment as I describe above but with one tube of blood that is frozen immediately and another one that is let to clot to obtain the serum: if you get similar results from the two tube in assessing one of the proteins you want to measure, your first tubes already collected may not be lost, and you can thank the mice ;)
You'll need to do a validation experiment. Mix ELISA standard into these samples in known amounts, see if the assay picks up the correct increase amounts, compared with pure standards. If it does, there should be nothing wrong with counting the results. (Explaining it to your reviewer for publication might be another matter...)
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