Table 1 Materials to generate autophagy reporter cell lines.
From: An improved method for high-throughput quantification of autophagy in mammalian cells
Product | Source | Product number |
|---|---|---|
Phusion high-fidelity DNA polymerase (2 U/µL) | Thermo Scientific | F530 |
dNTP Mix (10Â mM each) | Thermo Scientific | R0191 |
Dimethyl sulfoxide | Merck | 102952 |
Restriction Enzymes | New England Biolabs | Â |
Silica Bead DNA Gel Extraction Kit | Thermo Scientific | K0513 |
DNA Ligation Kit Ver.2.1 | Takara | 6022 |
Polyethylenimine, branched | Sigma-Aldrich | 408727 |
Opti-MEM I Reduced Serum Medium | Gibco | 31985047 |
Millex-HA Syringe Filter Unit, 0.45 µm, mixed cellulose esters, 33 mm, ethylene oxide sterilized | Merck | SLHA033SS |
Trypsin 0.05%/EDTA 0.02% in PBS, w/o: Ca and Mg | PAN-Biotech | P10-023100 |
Falcon Round-Bottom Tubes with Cell Strainer Cap, 5Â mL | Falcon | 38030 |
HEK293T cells | ATCC | CRL-3216 |
HeLa cells | ATCC | CCL-2 |
Jurkat cells | ATCC | TIB-152 |
THP-1 cells | ATCC | TIB-202 |
Media | ||
RPMI 1640 Medium | Gibco | 21875034 |
Gibco DMEM, high glucose, no glutamine | Gibco | 11500416 |
Fetal Bovine Serum, qualified, Brazil | Gibco | 10270106 |
Penicillin–Streptomycin, 10,000 U/ml Penicillin, 10 mg/ml Streptomycin | PAN-Biotech | P06-07050 |
L-Glutamine 200Â mM | PAN-Biotech | P04-80050 |
Verification | ||
Anti-GFP antibody | Abcam | ab290 |
Anti-LC3B antibody | Cell Signaling Technology | #2775 |
Anti-β-actin antibody | Invitrogen | AM4302 |
Paraformaldehyde solution 4% in PBS | Santa Cruz Biotechnology | sc-281692 |
Chloroquine diphosphate salt | Santa Cruz Biotechnology | sc-205629 |
InSolution Rapamycin | Merck | 553211 |
