1. Isolation of Mononuclear Cells from Umbilical Cord Blood
Prepare EDTA-PBS buffer- add 5 ml bovine serum albumin (BSA) stock solution to 95 ml rinsing buffer (1:20 dilution). Degas the buffer and keep the buffer on ice. IMPORTANT: Failure to degas the buffer may result in less than optimal results when isolating CD19+ B-cells because bubbles may block the isolation column.
Prepare 50 ml conical tubes for density gradient centrifugation. Determine the number of tubes required for processing the cord blood (1 tube can process 8 ml blood) and add 15 ml of Ficoll-Paque PLUS to each tube.
Dilute 8 ml of cord blood with 24 ml DPBS (1X) and carefully layer the diluted cord blood mixture on top of the Ficoll-Paque PLUS in each of the 50 ml conical tubes. Do not mix the blood and Ficoll-Paque PLUS. IMPORTANT: To avoid mixing of the cord blood and Ficoll-Paque PLUS, hold the tube at a 45 degree angle and layer the blood mixture slowly.
Centrifuge at 400 x g for 40 min at 20 °C. Mononuclear cells (MNC) will remain at the plasma-Ficoll-Paque PLUS interface whereas granulocytes and erythrocytes sediment due to higher density at the osmotic pressure of Ficoll-Paque PLUS. Label seven 5 ml round-bottom tubes to be used in Part 3 with the sample ID, date and the following:
Tube Label Purpose
1 Unstained Unstained cells for normalization during flow cytometry
2 7AAD To determine viability during flow cytometry
3 +++ Contains the cells that will be sorted
4 34+ To collect CD34+/CD19+ (late pro-B - early pre-BI) cells
5 45low To collect CD34-/CD19+/CD45low (pre-BI) cells
6 45med To collect CD34-/CD19+/CD45med (pre-BII) cells
7 45high To collect CD34-/CD19+/ CD45high (immature B) cells
Coat tubes 4, 5, 6, and 7 with 2% FBS and place all tubes on ice.
Aspirate the upper plasma layer carefully and avoid contact with the mononuclear cell layer. Using a 10 ml glass pipette, carefully transfer the mononuclear cell layer to a new 50 ml conical tube. Combine the mononuclear cells from three tubes together into a single 50 ml tube.
Fill the tube with PBS, mix gently and centrifuge at 300 x g for 10 min at 20 °C. Carefully aspirate the supernatant without disturbing the cell pellet. Repeat 1x. After the first wash, resuspend the pellets and transfer to a single 50 ml conical tube.
Gently resuspend the cell pellet in 200 μl of PBS. Remove 1 μl of the cell suspension, add it to 1 ml of PBS in a 1.5 ml microcentrifuge tube and set aside for counting (count cells after placing the 50 ml cell suspension in the centrifuge). Fill the tube with PBS and centrifuge at 200 x g for 15 min to remove platelets. Remove the supernatant completely without disturbing the cell pellet.
