I have four specific questions regarding RBC's isolation from blood?
Procedure-Procedure to isolate RBC's from whole blood?
Can i use ficol method to isolate RBC's? my real concern for this procedure is, if ficol interferes with RBC's or not?
Counting- Should i use trypan blue to count RBCs by hemocytometer or i can count them without trypan staining.
Number-In general what are the number of RBC's in 1ml of blood after isolation?
Time- How long (may be 4-5 hr) i can keep RBC's in 1XPBS to perform experiment?
You do not need complicated procedures. Centrifuge the whole blood at 2000 for 5 min. With a pipette, separate plasma and the buffycoat layer from red blood cells. Add saline + 5 mM glucose (at least 1:1) and gently invert then times the tube to mix. Centrifuge again at 2000g and repeat this step other 3 times. Just a tip: to easily the buffycoat layer you can mix it with the tip of the pipette (aftter plasma removl) with the red cells below and remove both. Doing this you lose also a part of red cells but you are sure that you have liminated all others. For counting you do not need trypan blue staining, after isoltation packed red cells are >10^7 uL. Usually if stored at 4°C in saline containing 5 mM glucose can be used for 3-4 days but it obviously depends of what kind of experiments you want to perform.
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