Vishwanath Somashekar

Hi all,
I need to make RBC ghosts for running my experiments. I basically need to get the hemoglobin out of the rbcs and reseal them. As have the hemoglobin puts a limitation on the hematocrit I can use for my experiments. I would really appreciate if one of you could give me a detailed protocol. I am a mechanical engineer and that makes these processes a little more complicated than they are.

For example, I followed protocol from Cha and Beinssinger (1996b). It requires that the rbc's be cleaned in bps (pH 7.4) three times at 4500g for 15 mins (which I did). However, even after 3 washes, i saw a light red supernatant.

Then I need to suspend this in 40 volumes of pbs (pH 8) overnight. When I came in this morning, i see the cells settled, and a pink liquid on the top. When I centrifuged, was hoping to see no red colored cells pelleted. Instead, I still see the rbc pelleted at the bottom of the tube bright red in color. Not sure if I did something wrong. I tried to google extensively to see if there are pictures/video of the procedure, and unfortunately, I couldn't' find any.

I would really appreciate if any of you can help  me out.

Thanks a bunch in advance,
Vishwa