CD31-FITC and anti-CD45-FITC antibodies by adding 10 μL
of each antibody and incubating at 4
C for 15 min.
3. Aspirate unbound antibodies after adding 2 mL of column
buffer and subsequent centrifugation at 300 Â g for 10 min.
4. Resuspend the cell pellet in 90 μL of column buffer, add 10 μL
of MACS anti-FITC magnetic microbeads, and incubate at
4
C for 15 min.
5. Aspirate unbound microbeads after adding 2 mL of column
buffer and subsequent centrifugation at 300 Â g for 10 min at
4
C.
6. Resuspend the cell pellet in 500 μL of column buffer, and place
a MACS LD column on MidiMACS separator for each cell
pellet.
7. Prepare each column by washing with 2 mL column buffer.
8. Pipette each cell suspension into individual columns, and collect unlabeled cells flowing through the columns into 15-mL
tubes.
9. Add 1 mL column buffer to each column to wash out all
unlabeled cells.
3.3 Controlled-Rate
Freezing
of Human ASCs
1. Prepare cryopreservation media in advance. When protected
from light and stored at 4
C, cryopreservation media are good
for 2 weeks. Therefore, preparation of cryopreservation media
in small volumes is recommended.
2. Enter the following ASC cooling program into the controlled
rate freezer (see Note 7):
(a) Step 1: Rapid cooling (10
C/min or 20
C/min) from
RT to the start temperature (i.e., 0
C).
(b) Step 2: Hold at 0
C until samples are introduced and the
program is activated.
(c) Step 3: Cooling to À7
C at 2
C/min.
(d) Step 4: Soak time for 2 min and subsequent manual seeding of extracellular ice at À7
C.
(e) Step 5: Holding at À7
C for 10 min.
(f) Step 6: Cooling to À70
C at 1
C/min and holding at
À70
C until transferring samples to liquid nitrogen.
3. Label cryovials or irradiated sterile 0.5-cc straws under the
hood (see Note 8).
4. Open liquid nitrogen supply valve, turn on the controlled-rate
freezer, and run the ASC cryopreservation program to precool
the chamber to the start temperature.
562
Melany Lo ´ pez and Ali Eroglu
of each antibody and incubating at 4
C for 15 min.
3. Aspirate unbound antibodies after adding 2 mL of column
buffer and subsequent centrifugation at 300 Â g for 10 min.
4. Resuspend the cell pellet in 90 μL of column buffer, add 10 μL
of MACS anti-FITC magnetic microbeads, and incubate at
4
C for 15 min.
5. Aspirate unbound microbeads after adding 2 mL of column
buffer and subsequent centrifugation at 300 Â g for 10 min at
4
C.
6. Resuspend the cell pellet in 500 μL of column buffer, and place
a MACS LD column on MidiMACS separator for each cell
pellet.
7. Prepare each column by washing with 2 mL column buffer.
8. Pipette each cell suspension into individual columns, and collect unlabeled cells flowing through the columns into 15-mL
tubes.
9. Add 1 mL column buffer to each column to wash out all
unlabeled cells.
3.3 Controlled-Rate
Freezing
of Human ASCs
1. Prepare cryopreservation media in advance. When protected
from light and stored at 4
C, cryopreservation media are good
for 2 weeks. Therefore, preparation of cryopreservation media
in small volumes is recommended.
2. Enter the following ASC cooling program into the controlled
rate freezer (see Note 7):
(a) Step 1: Rapid cooling (10
C/min or 20
C/min) from
RT to the start temperature (i.e., 0
C).
(b) Step 2: Hold at 0
C until samples are introduced and the
program is activated.
(c) Step 3: Cooling to À7
C at 2
C/min.
(d) Step 4: Soak time for 2 min and subsequent manual seeding of extracellular ice at À7
C.
(e) Step 5: Holding at À7
C for 10 min.
(f) Step 6: Cooling to À70
C at 1
C/min and holding at
À70
C until transferring samples to liquid nitrogen.
3. Label cryovials or irradiated sterile 0.5-cc straws under the
hood (see Note 8).
4. Open liquid nitrogen supply valve, turn on the controlled-rate
freezer, and run the ASC cryopreservation program to precool
the chamber to the start temperature.
562
Melany Lo ´ pez and Ali Eroglu