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Page Title:
APPENDIX II. LABORATORY PROTOCOLS FOR P450RGS DIOXIN SCREENING ASSAY |
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 ERDC TN-DOER-C10
March 2000
APPENDIX II
LABORATORY PROTOCOLS FOR P450RGS DIOXIN SCREENING ASSAY
Protocol I. Cell Culture and Reagent Preparation.
I. MATERIALS AND EQUIPMENT.
Vertical laminar flow hood.
37 C CO2 incubator.
Countertop centrifuge.
Autoclave.
Ultrapure water.
Sterile filters (0.1 m), VacuCap 90 (Gelman Sciences #09730265B).
Sterile, disposable 5- and 10-mL and pasteur pipets (9 in., autoclaved).
Sterile T-75 (75-cm2 growth surface area) culture flasks.
Culture tubes (10 75, FisherBrand #14-961-25).
Sterile syringes and 0.22-m syringe filters.
Squirt bottle of 70 percent reagent alcohol.
Parafilm.
Waterbath at 37 C.
Alcohol bath.
Aspirator.
Rechargeable portable multichannel pipettors (8-channel) and tips.
Inverted microscope.
Isopropanol freezing chamber .
Liquid nitrogen Dewar, -195 C.
101L human hepatoma cells
II.
REAGENTS.
A.
PRODUCT INFORMATION.
Minimum Essential Medium (MEM), GIBCO BRL #51200-038.
Versene (1x, 0.2 g EDTA.4Na), GIBCO BRL #15040-066.
Fetal Bovine Serum (FBS), GIBCO BRL #16000-044.
L-Glutamine (200 mM, 29.2 mg/mL), GIBCO BRL #25030-081.
Geneticin (G418), GIBCO BRL #11811-031.
Phosphate Buffered Saline (PBS), GIBCO BRL #14190-144.
Sodium Pyruvate (11.0 mg/mL), SIGMA #S-8636.
Trypsin (with phenol red, 2.5 mg/mL), SIGMA #T-4424.
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