Hi all
I'm trying to perform the procedure of sickling-induction in-vitro of peripheral red blood cells from sickle cell Townes mice, and I'm using 2% sodium metabisulfite. Unfortunately the protocol doesn't seem to work perfectly, as sometimes I see quite a lot of sickling, yet other times I can't really see much or any at all. I always compare a sickle cell mouse with a normal human Hb mouse. I obtain blood from saphenous bleed; using a heparinized capillary tube I transfer the blood to a microtube containing 3-5 uL of heparin. The 2% sodium metabisulfite is freshly made within 30-60 minutes, resuspended in PBS. I mix 10 uL of sodium metabisulfite solution with 10 uL of heparinized blood, and then transfer 10 uL of the mix into a slide, which I cover and then seal with transparent nail polish. I incubate my slides for 45-60 minutes at 37C 5% CO2. I look at the slides under phase contrast microscopy.
Does anyone have a good idea how to make more consistent my sickling effect?
Thanks in advance!
My suggestion is to follow the method described in practical haematology by Dacie & Levis (now edited by B.Bain). They use Sodium dithionite (Na2S2O4), which is a stronger reducing agent than Sodium metabisulphite (Na2S2O5). There it is mixed with Na2HPO4. Kindly go through the details of the test in the book. Another suggestion is to use whole anti-coagulated blood in very small quantity mixed with a large drop of test solution to get a very dilute red cell suspension and seal under a cover slip, so that under high power of microscope you can see the red cells well dispersed and see individual red cell for morphological change. If you try this kindly let me know the result and whether it gives better results or not.