Hi everyone.
This is my first time working with the mouse blood collection. I already read so many protocols but I keep failing.
I collect the blood from saphenous vein of the 22 gram C3H mouse and instead of using heparinized tube, I use 50 microliter of heparin solution ( simply dilute 1 mg sodium heparin in 4 ml distilled water, that will contain around 2-5 IU each 50 microliter).
I successfully prevent the coagulation, but during the separation of the plasma I get hemolyzed sample (the colour of the plasma still red). I use 13000 rpm, 4 degree celcius, 15 minutes for centrifugation and try to put directly in the ice after collection.
I am afraid I make mistake during preparation of heparin solution.
So I really need your suggestion to improved my protocol.
Thank you so much
The use of distilled water instead of normal saline may be one possible reason. Normal saline is isotonic; water is hypotonic, and can cause cell lysis due to the difference in osmotic pressure. Try using normal saline instead of distilled water and see if it makes a difference.
Dear Mr. Greg Cote.
Thank you so much Sir.
It does work really well with normal saline.
I really appreciate.
thank you so much.
It depend what you want to mesure after blood collection.
I think EDTA, by complexing Ca and so inhibite coagulation, is the best way
For plasma separation, in a 1.5 ml ependorf mirotube 25 Microliter (0.4 IU) diluted heparin sodium (add 1 ml PBS 1X to 33 IU heparin tube) used for 7- 10 drop blood from the tail mouse. the attached is heparin tube information. 2- leave the tube until 30 min
3- centrifuge 1200 rpm 10 min
You can separate plasma using tubes containing sodium EDTA anticoagulant, centrifuge immediately after collecting blood. Separate plasma by centrifugation at 1,500 x g for 10 minutes using a refrigerated centrifuge at 4°C.
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