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.
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