Separation of normal CD34+ cells from fresh pheresis of mobilized stem cells

Separation of normal CD34+ cells from fresh pheresis of mobilized stem cells

1.      Resuspend sample up to 100 mL of MACS buffer (see recipe below). Aliquot to two 50-mL conical tubes. Centrifuge for 10 minutes at 1000 rpm to "soft spin" the pellet. A soft spin keeps the platelets, which are concentrated in pheresis samples, in the supernatant. Platelets cause major problems with the staining of the sample as well as the running of the sample through the magnetic column. The supernantant must be aspirated, not poured off, since the pellet is loose.

2.      If the supernatant from the soft spin is still relatively cloudy, the soft spin may be repeated.

3.      ACD-A changes the density of the cells in the pheresis sample so that granulocytes will stay at the interphase of the Ficoll. Although pheresis samples have a high concentration of granulocytes, it is more important to remove the platelets before they activate. An alternative protocol may be that the sample is initially suspended in buffer without ACD-A and then Ficolled. The mononuclear layer may then be resuspended in buffer with ACD-A, and the soft spin performed. We have found it best to just leave the granulocytes and adjust concentration of the antibody (follows).

4.      Count the total number of cells. Combine sample into one 50-mL conical tube. Miltenyi lists antibody amount according to the total number of cells; however, this may be adjusted according to the estimate of number of cells positive for the sorting parameter. Since pheresis has between 1-10% CD34+ cells, we usually use 50% of the recommended amount of antibody. If a Ficoll is not performed, this may be reduced to ? the amount of antibody, but the buffer should not go below ?, and the incubation time should be extended to 30 minutes. Add ? the amount of buffer recommended by Miltenyi. Add ? the amount of reagent A1, shake gently. Add ? the recommended amount of reagent A2, shake gently. Incubate in the refrigerator for 15 minutes, gently shaking the sample periodically.

5.      Wash the sample two times with 50 mL of MACS buffer.

6.      Resuspend the sample in _ the recommended amount of buffer, and _ the amount of reagent B, shake gently. Incubate in the refrigerator for 15 minutes, gently shaking the sample periodically.

7.      Wash the sample one time with 50 mL of MACS buffer.

8.      Resuspend the sample in at least 10 mL degassed (see Note) MACS buffer for 1 x 109 total cells, or up to 20 mL for 2 x 109 total cells. Run sample over a VS positive selection column.

9.      Wash 2X with 3 mL of degassed buffer.

10.   Attach stop cock and syringe to bottom of VS column. Remove column from magnet. Backflush column with 6 mL of degassed buffer. Replace column in magnet.

11.   Remove stop cock. Allow buffer to flow through column. Wash 2X with 3 mL of buffer.

12.   Remove column from magnet. Add 6 mL buffer to column and allow to run through. Add 6 mL buffer to column and plunge the column.

13.   Count the total number of cells collected from each fraction to calculate the recovery of the separation.

14.   Perform flow cytometry on the collected fractions to assess sample purity with CD45-FITC and CD34-PE (Becton Dickinson).

MACS buffer

Hank's Balanced Saline Solution (HBSS) -Ca+2, -Mg+2

0.5% BSA

0.6% Anticoagulant Citrate Dextrose- Formula A (ACDA) (Baxter)

Filter sterilize and store at 4°C

Notes: It is very important when running the magnetic column that only degassed MACS buffer be used. To degas the buffer, place 100 mL of buffer in a 150-mL bottle. Place a rubber stopper attached to a vacuum line over the mouth of the bottle. Turn vacuum on. Allow the buffer to degas at room temperature for at least 30 minutes. Replace cap on bottle and refrigerate buffer until cold. Use buffer as directed.