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 ERDC TN-DOER-C30
February 2003
The following times are required to complete each daily task using the 96-well plates:
Day
Time, hr
1
1.0
2
1.0
4
1.0
Solution preparation/equipment setup
1.0
Total
4.0
II.
Day 1 (PLATING CELLS)
A.
MATERIALS AND EQUIPMENT
MEM
PBS
Trypsin-EDTA (1x) - 1 aliquot
Trypan Blue (0.05 percent) - one aliquot
Multichannel motorized pipettor (250 L)
Sterile pipet tips (250 L)
Pasteur pipets (9", autoclaved)
Hemacytometer and cover slip
Inverted microscope
50-mL sterile centrifuge tube
50-mL sterile solution basins
Water bath set at 37 C
B.
PROCEDURE (use sterile technique)
1) Wipe down the working surfaces of the laminar flow with 70 percent alcohol.
2) Turn on the ultraviolet light and blower of the laminar flow hood. Allow 15 min
for equilibration.
3) Turn on 37 C water bath.
4) Place supplemented MEM and one aliquot each of Trypan Blue and trypsin-EDTA
in the water bath for about 30 min.
5) Trypsinize one confluent (T-75) flask of HepG2-EcR cells with 3-mL trypsin-EDTA
each as described in "SUBCULTURING CELLS." A confluent flask contains
approximately 10 to 13 106 cells. Thus, one confluent flask of cells should be
sufficient for seeding two 96-well plates at 40,000 cells/200 L/well.
6) Transfer the cell suspension (approximately 13 mL) from the T-75 flask into a
sterile 50-mL centrifuge tube.
7) Pipet 200 L of cell suspension into the 1.800 mL of Trypan Blue solution.
Centrifuge the remaining cell suspension at 1,000 rpm's for 5 min.
8) Count the cells using a hemacytometer to determine the number of cells/mL of
cultured stock (described in "COUNTING CELLS").
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