| Basic Protocol 1: Expression of Cloned cDNA Transfected into Mammalian Cells Materials -
LBA medium: LB medium ( appendix 2A ) containing appropriate antibiotics -
DMSO or glycerol stocks of cDNA pools (see protocol 3 or protocol 42 ) -
TE buffer, pH 7.5 ( appendix 2A ) -
Phosphate‐buffered saline (PBS; appendix 2A ) -
Trypsinization solution: 0.25% (w/v) trypsin/0.02% (w/v) EDTA -
Complete DMEM/10% FBS, complete DMEM/20% FBS, and complete DMEM without serum ( appendix 2A ) -
COS‐1, COS‐7, or HEK‐293 cells (ATCC# CRL 1650, CRL 1651, and CRL 1573, respectively) -
250‐ml flasks, sterile -
Shaking incubator, 37°C -
Single‐chamber tissue culture slides, 6‐well tissue culture plates, or 10‐cm tissue culture dishes, sterile -
Additional reagents and equipment for bioassay of transfected cells (see Chapter 7), isolation and purification of plasmid DNA, counting cells using a hemacytometer, DNA quantitating, calcium phosphate– or lipid‐mediated transfection, and transfection by electroporation (see CPMB UNITS , , , & , CPMB APPENDICES & , and appendix 1A in this manual) NOTE: All solutions and equipment coming into contact with live cells must be sterile and proper sterile technique should be used accordingly. NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Basic Protocol 2: Recombinant Vaccinia Virus Infection and Liposome‐Mediated Transfection of Mammalian Cells Materials -
CV‐1 or HeLa cells (ATCC #CCL 70 and CCL 2, respectively) -
Complete DMEM/10% FBS, complete DMEM/20% FBS, and complete DMEM without serum ( appendix 2A ) -
Recombinant T7 (Fuerst et al., ) or SP6 (Usdin et al., ) vaccinia virus stock (available from B. Moss, NIH) -
Purified DNA from cDNA pools (see protocol 1 , steps and ) -
LipofectACE (Life Technologies) -
Bleach -
10‐cm tissue culture dishes, single‐chamber culture slides, or 6‐well tissue culture plates (optional) -
Bath sonicator with ice water -
24‐well polystyrene tissue culture plates or polystyrene tubes NOTE: All solutions and equipment coming into contact with live cells must be sterile and proper sterile technique should be used accordingly. NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Support Protocol 1: Preparing cDNA Pools by Culture in Antibiotic Selection Medium Materials -
DMSO or glycerol stock cDNA library from appropriate cells or tissue transformed into E. coli or packaged into a phage (e.g., λ ZAP , Stratagene) -
LBA medium: LB medium ( appendix 2A ) containing appropriate antibiotics -
LBA plates: 10‐cm LB agar plates ( appendix 2A ) containing appropriate antibiotics -
Glycerol stock buffer (see recipe ) Support Protocol 2: Preparing cDNA Pools by Plating onto Antibiotic Selection Plates Materials -
DMSO or glycerol stock cDNA library from appropriate cells or tissue transformed into E. coli or packaged into a phage (e.g., λ ZAP , Stratagene) -
LBA plates: 10‐ and 15‐cm LB agar plates ( appendix 2A ) containing appropriate antibiotics -
LBA medium: LB medium ( appendix 2A ) containing appropriate antibiotics -
Glycerol stock buffer (see recipe ) Support Protocol 3: Using Autoradiography to Detect Gene Expression Materials -
Physiological buffer: HEPES‐buffered Krebs‐Ringer buffer (see recipe ) or phosphate‐buffered saline (PBS; appendix 2A ) -
Cultures of adherent transformed cells in 10‐cm tissue culture plates (see protocol 1 or protocol 2 ) -
Glogos luminescent stickers (Stratagene) -
SDS/EDTA: 0.6% (w/v) SDS/10 mM EDTA -
5 M NaCl -
recipeTris‐buffered phenol ( appendix 2A ) -
20 mg/ml glycogen -
100% ethanol -
recipe3 M sodium acetate ( appendix 2A ) -
TE buffer, pH 7.5 ( appendix 2A ) -
Electroporation‐competent E. coli cells -
LBA medium: LB medium ( appendix 2A ) supplemented with appropriate antibiotics -
Double‐sided tape -
X‐ray film: XAR or Beta‐max MR (Kodak) -
Film cassettes, preferably large -
Additional reagents and equipment for binding or transport assays for gene expression (e.g., see Chapter 7), electroporation of E. coli ( appendix 1E ), preparation of glycerol stock (see protocol 3 ), and transfection of COS cells (see protocol 1 ) Support Protocol 4: Using Nuclear Emulsion–Coated Chamber Slides to Detect Gene Expression Materials -
Physiological buffer: HEPES‐buffered Krebs‐Ringer (see recipe ) or phosphate‐buffered saline (PBS; appendix 2A ) -
Transformed cells plated on single‐chamber tissue culture slides (see protocol 1 or protocol 2 ) -
Radioligand fixative (see Table 4.8.1 ) -
PBS ( appendix 2A ) -
NTB‐2 nuclear track emulsion (Kodak or equivalent) -
Dektol or D19 developer and fixer (Kodak) -
Counterstain: e.g., 1% (w/v) Giemsa or 0.05% (w/v) nuclear fast red -
Low‐viscosity Cytoseal (e.g., Fisher) -
Slide rack -
Additional reagents and equipment for performing binding or transport assay (see Chapter 7) Table 4.8.1 Materials Radioligand and Radiosubstrate Fixatives a Radioligand and Radiosubstrate Fixatives
| | Fixative | | 4% (w/v) formaldehyde/PBS b | | Acetone | | Methanol | | 1% (v/v) acrolein c /2.5% glutaraldehyde d /0.1 M cacodylate buffer, pH 6.4 e | | 1% (v/v) acrolein c /2.5% glutaraldehyde/PBS, pH 7.4 b | | 10% (v/v) acrolein c /0.1 M cacodylate buffer e | | 1% (v/v) glutaraldehyde d /PBS, pH 7.4 b | | 1% (v/v) glutaraldehyde d /0.1 M cacodylate buffer, pH 6.4 e | | 5% (v/v) glutaraldehyde/0.1 M cacodylate buffer, pH 6.4 e | | 1% (v/v) glutaraldehyde d /4% (w/v) formaldehyde/5% (v/v) saturated picric acid solution | | 1% (w/v) osmium tetroxide l /0.1 M cacodylate buffer, pH 6.4 e | | 1% (w/v) potassium permanganate/0.1 M cacodylate buffer, pH 6.4 e | | 3% (w/v) potassium permanganate/acetate buffer e | |
| a These solutions represent a range of possibilities as a starting point for determining what might work best for the radioligand of interest. Selection of the optimal fixative will depend on the chemical structure of the ligand or substrate in use. For instance, acrolein worked well for the neurotransmitter 5‐hydroxytryptamine since acrolein reacts with the compound's indole nitrogen. For further information concerning fixatives, consult McManus and Mowry ( ). b See recipe in appendix 2A . c CAUTION: Acrolein is tear gas and should only be used in a hood. d Electon microscopy grade, stored at 4°C. e See recipe in Reagents and Solutions . | Support Protocol 5: Transport Activity Assay to Detect and Quantify Activity of Expressed Genes Materials -
Transfected cells grown in 6‐well tissue culture plates (see protocol 1 or protocol 22 ) -
Uptake buffer (see recipe ), 37°C and ice cold -
Radioligand working solution at 7‐fold desired final concentration (see unit 7.9 ) -
Inhibitor of transport diluted in uptake buffer to 7‐fold desired final concentration (see unit 7.9 ) -
0.5 N NaOH or 1% (w/v) SDS -
Scintillation fluid -
Micropipettor with polypropylene tips
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