206
BURT G. FEUERSTEIN et al.
Metaphase
production
Making metaphase
spreads
1. Aspirate complete medium and gently wash cell layer with 8 ml 37°C
Phosphate buffered saline, Ca++ and Mg++ free.
2. Place 3 ml 0.25% trypsin into each flask. Cap the flasks securely and
place horizontally in incubator for 5 min.
3. Neutralize the trypsin by adding 6 ml complete medium; repeatedly
aspirate and squirt the medium to detach the cells from the flask surface.
4. Split the cells from one T-25 into one T-75 flask and top off with media
to 8 ml. Cap the flasks and return to the incubator.
5. Examine cells after about 4 h. They should be 30%-40% confluent. Reexamine cells periodically afterI8 h.
6. When the culture has reached 70-80% confluence (usually about 24 h),
treat with 107
M methotrexate. For a culture that doubles every 24 h,
treat the cells 17 h.
7. Release cells with lO- s M thymidine. Incubate 2.5 h.
8. Add 0.02 Ilglml colcemid. Incubate cells 30 min.
9. Wash cells gently with phosphate buffered saline (37°C). Gently swirl
and aspirate.
10. Add 2 ml 0.25% trypsin prewarmed to 37°C and incubate 5 min at 37°C.
11. Add 4 ml complete medium and wash the cells off the side of the flask.
Place the cells into a 15-ml tube.
1. Centrifuge cells at 1000 rpm for 5 min. Remove supernatant, leaving
about 100 III medium.
2. Add 50 III 0.075 M KCI (prewarmed to 37°C) to the tube and mix cells
very gently with a pipette.
3. Slowly add three more 50 III volumes of 0.075 M KCI to the cells, all the
while mixing gently with a pipette.
4. Increase the volume added to 100 Ill, adding three volumes while mixing gently. Repeat with 200 III volumes, then 300 Ill, 400 III and 500 III
volumes. Bring the cell suspension to a total volume of 7 ml, and incubate at 37°C for 15 min.
5. Add 10 drops of fresh Carnoy's solution. Centrifuge cells at 1000 rpm
for 5 min. Pour off supernatant.
BURT G. FEUERSTEIN et al.
Metaphase
production
Making metaphase
spreads
1. Aspirate complete medium and gently wash cell layer with 8 ml 37°C
Phosphate buffered saline, Ca++ and Mg++ free.
2. Place 3 ml 0.25% trypsin into each flask. Cap the flasks securely and
place horizontally in incubator for 5 min.
3. Neutralize the trypsin by adding 6 ml complete medium; repeatedly
aspirate and squirt the medium to detach the cells from the flask surface.
4. Split the cells from one T-25 into one T-75 flask and top off with media
to 8 ml. Cap the flasks and return to the incubator.
5. Examine cells after about 4 h. They should be 30%-40% confluent. Reexamine cells periodically afterI8 h.
6. When the culture has reached 70-80% confluence (usually about 24 h),
treat with 107
M methotrexate. For a culture that doubles every 24 h,
treat the cells 17 h.
7. Release cells with lO- s M thymidine. Incubate 2.5 h.
8. Add 0.02 Ilglml colcemid. Incubate cells 30 min.
9. Wash cells gently with phosphate buffered saline (37°C). Gently swirl
and aspirate.
10. Add 2 ml 0.25% trypsin prewarmed to 37°C and incubate 5 min at 37°C.
11. Add 4 ml complete medium and wash the cells off the side of the flask.
Place the cells into a 15-ml tube.
1. Centrifuge cells at 1000 rpm for 5 min. Remove supernatant, leaving
about 100 III medium.
2. Add 50 III 0.075 M KCI (prewarmed to 37°C) to the tube and mix cells
very gently with a pipette.
3. Slowly add three more 50 III volumes of 0.075 M KCI to the cells, all the
while mixing gently with a pipette.
4. Increase the volume added to 100 Ill, adding three volumes while mixing gently. Repeat with 200 III volumes, then 300 Ill, 400 III and 500 III
volumes. Bring the cell suspension to a total volume of 7 ml, and incubate at 37°C for 15 min.
5. Add 10 drops of fresh Carnoy's solution. Centrifuge cells at 1000 rpm
for 5 min. Pour off supernatant.