removing rbc cells for cell purification - (Aug/04/2007 )

I was going to lyse the rbc with 10X DPBS or 0mosm/L water and then run the harvested wbc through a 1.077g/ml density gradient to harvest the mnc. I need to know if these solutions or another chemical to remove the rbc will be safe and give me a high yield of mnc, if the monocytes and b-cells are already activated and adhering to each other. This is why I have to remove the rbc first. Any ideas?
Thank you,
Dave
P.S. My tele is 813-971-6507, and I only check my messages once per week.

---

I have been using ACK to lyse RBCs when I need to but with Ficoll Hq Method, there is no much contamination with RBC that I need to do RBC lysis.

(sorry, can't call U; can't make int call and U have not disclosed location either)

---

I am in Florida, U.S. I cannot initially use any density gradient (my nycodenz) because I am working with bad blood. The red blood cells keep pushing out the adhering b-cells and monos from the 1.077g/ml gradient.
thank you,
Dave

---

I used 1/10 PBS to lyse RBCs just 10 second (not too long to destroy MNCs), then add 0.1 Vol of 10x PBS to restore Osmos.

---

In the past we have used the following method to remove by lysis RBC's:

Resuspend your WBC/RBC suspension in ice cold lysis buffer (0.82% NH4Cl containing 5mM KCl brought to pH 7.4 with 4.4% NaHCO3). This procedures lyses all RBC's during 10 minute incubation on ice.

We were after a pure population of neutraphils for Degranulation and Chemotaxis assays. This procedure had no significant effect on the neutraphils, all were biochemically actiev.

Rhombus

---

---