Appendix I. LABORATORY PROTOCOLS FOR HepG2-EcR SCREENING ASSAY

ERDC TN-DOER-C30

February 2003

Appendix I

LABORATORY PROTOCOLS

FOR HepG2-EcR SCREENING ASSAY

Protocol I. Cell Culture and Reagent Preparation

MATERIALS AND EQUIPMENT

Vertical laminar flow hood

37 C CO2 incubator

Countertop Centrifuge

Autoclave

Ultra-pure water

Sterile filters (0.1 m), VacuCap 90 (Gelman Sciences #09730265B)

Sterile, disposable 5, 10 mL, and pasteur pipets (9", autoclaved)

Sterile T-75 (75 cm2 growth surface area) culture flasks

Sterile syringes and 0.22-m syringe filters

Squirt bottle of 70 percent reagent alcohol

Parafilm

Water bath @ 37 C

Alcohol bath

Aspirator

Rechargeable portable pipettors and tips

Inverted microscope

Isopropanol freezing chamber

Liquid nitrogen Dewar @ -195 C

HepG2-EcR (recombinant human hepatoma) cells

II.

REAGENTS

A. PRODUCT INFORMATION

Minimum Essential Medium (MEM), GIBCO BRL #51200-038

Fetal Bovine Serum (FBS), GIBCO BRL #16000-044

L-Glutamine (200 mM, 29.2 mg/ml), GIBCO BRL #25030-081

Hygromycin B (100 mg/ml), InVitrogen #55-0280

Zeocin (100 mg/ml), InVitrogen #R250-01

Phosphate Buffered Saline (PBS), GIBCO BRL #14190-144

Trypsin-EDTA (0.5-percent Trypsin, 0.53-mM EDTA), 10x, GIBCO BRL #15400-054

Trypan Blue (0.4 percent), GIBCO BRL# 15250-061

Dimethyl Sulfoxide (DMSO), SIGMA #D-2650

Cell culture handling and reagent preparation throughout the assay should be carried out only under

a sterilized laminar hood using aseptic techniques. Only the last part of the HepG2-EcR assay

("Day 4 (Assay Takedown)" in the "Performance of the Assay" section) need not be sterile.