1.5 × 106 control or TMEM16A overexpressing OSC19 or UM-SCC-1 cells were implanted in 100μL of Matrigel subcutaneously in nude mice. In OSC19 xenograft, each mouse had control or TMEM16A overexpressing tumors on either flank and were treated with vehicle control (60μL sterile water; n = 11) or cisplatin (3mg/kg in sterile water; n = 10). At the end point mice were euthanized in accordance with University of Pittsburgh guidelines for animal use. Tumors were removed and weights were recorded. Lysates of tumors were prepared and protein was estimated. Equal amount of protein was separated in 8% gels and detected in western blots analyses with the indicated antibodies in LiCor imaging system (LiCor Odyssey Classic). All experiments performed in mice were conducted with approval from the University of Pittsburgh Institutional Animal Care and Use Committee.
Licor odyssey classic
Manufactured by LI COR
The LiCor Odyssey Classic is a near-infrared fluorescence imaging system designed for qualitative and quantitative analysis of proteins and other biomolecules. It provides high sensitivity and resolution for a wide range of applications, including Western blotting, protein array analysis, and cell-based assays.
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Lab products found in correlation
Blocking solution, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Irdyer 800cw, by LI COR (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Complete mini protease inhibitor, by Roche (1 mentions)
Chemiluminescence hrp substrate kit, by Merck Group (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Vero cell, by American Type Culture Collection (1 mentions)
7 protocols using licor odyssey classic
1
TMEM16A Overexpression in Xenograft Tumors
2
Zika and Dengue Virus Neutralization Assay
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PRNT was performed on acute-phase sera to detect neutralization antibody to ZIKV as reported previously (47 (link)). For late-convalescent-phase sera, a previously described microneutralization test was performed (48 (link)). Briefly, flat-bottom 96-well plates were seeded with Vero cells (3 × 104 cells per well) 24 h prior to infection. Fourfold serial dilutions of serum (starting from 1:10) were mixed with 50 focus-forming units of DENV1 (Hawaii strain), DENV2 (NGC strain), DENV3 (CH53489), DENV4 (H241 strain), or ZIKV (PRVABC59 strain) at 37°C for 1 h. The mixtures were added to each well followed by incubation for 48 h (except 70 h for DENV1), removal of medium, and fixation as described previously (46 (link)). After adding a murine monoclonal antibody (MAb) 4G2 and secondary antibody mixture (IRDye 800CW-conjugated goat anti-mouse IgG at 1:10,000 and DRAQ5 fluorescent probe at 1:10,000), the signal (800 nm/700 nm fluorescence) was detected by the use of a Li-Cor Odyssey Classic imaging system (Li-Cor Biosciences) and analyzed using Image Studio software to determine percent neutralization at different concentrations and NT90 as described previously (46 (link), 48 (link)).
3
Protein Isolation and Western Blot
4
Subcutaneous Tumor Growth Assay
5
Western Blot Analysis of EGFR Expression
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Three to four million cells were lysed in modified radio immunoprecipitation assay (RIPA) buffer (25 mM Tris HCl pH 7.6, 151.5 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1x Complete mini protease inhibitor (Roche, Indianapolis, IN), 1 mM PMSF) for 40 minutes on ice. The lysates were clarified by centrifugation (15 minutes at 14000 rpm). 30–40 μg of lysate was resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon, Millipore, Billerica, MA). Membranes were blocked with either 5% skim milk diluted in PBS-Tween (0.1%) or Odyssey blocking buffer (Licor Biosciences, Lincoln, NE) then incubated with anti-EGFR, anti-β-actin or anti-α-tubulin diluted in blocking buffer at 4°C overnight. The filters were incubated with secondary antibodies conjugated to horse radish peroxidase and the immunocomplexes detected with the Chemiluminescence HRP Substrate kit (Millipore) or incubated with IRDye conjugated secondary antibodies and the immunocomplexes detected by fluorescence using the Licor Odyssey Classic (Licor Biosciences).
6
Quantitative Dengue/Zika Neutralization Assay
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Flat-bottom 96-well plates were seeded with Vero cells (3 × 104 cells per well) (American Type Culture Collection, USA) 24 h prior to infection. Four-fold serial dilutions of serum or plasma (starting from1:10) were mixed with 50 focus-forming units of DENV1 (Hawaii strain), DENV2 (NGC strain), DENV3 (CH53489 strain), DENV4 (H241 strain), or ZIKV (PRVABC59 strain) at 37 °C for 1 h. The mixtures were added to each well followed by incubation for 48 − 70 h, removal of medium, and fixation as described previously [34 (link), 37 (link)]. After adding the mouse mAb 4G2 and secondary antibody mixture (IRDye® 800CW-conjugated goat anti-mouse IgG at 1:10000 and DRAQ5™ Fluorescent Probe at 1:10000), the signal (800 nm/700 nm fluorescence) was detected by Li Cor Odyssey classic (LiCor Biosciences) and analyzed by Image Studio software to determine percent neutralization at different concentrations and NT90 [34 (link)].
7
Multiplex Serological Assay for Flavivirus Detection
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Uninfected (mock) Vero cells and Vero cells infected with DENV1 (Hawaii strain), DENV2 (NGC strain), DENV4 (H241 strain), ZIKV (PRVABC59 strain), WNV (NY99 strain) or YFV (17D vaccine strain) were lysed with NP-40 lysis buffer (1% NP-40, 50 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, and 1 mM Na3VO4) when 50% of cells were found to have cytopathic effects. The cell lysates (1−5 µL per well) were loaded into two half-gels (seven wells each) and subjected to SDS-12% polyacrylamide gel electrophoresis under non-reducing condition (2% SDS, 0.5 M Tris pH 6.8, 20% glycerol, 0.001% bromophenol blue, final) [24 (link),40 (link)], followed by transfer to nitrocellulose membrane (Trans-Blot Turbo RTA Midi Transfer Kit, BioRad), hybridization with human serum/plasma samples (1:200 dilution) or mouse monoclonal antibody (mAb) and secondary antibody (IRDye® 800CW-conjugated goat anti-human IgG at 1:10,000). The signals were detected by Li Cor Odyssey classic (LiCor Biosciences) and analyzed by Image Studio software with both short and long exposures [40 (link),46 (link)]. Each gel was read independently by two researchers with the results summarized in Table S2. To test the stability of the assay, the half-membranes after blocking step were stored in the −20°C freezer until use for hybridization to serum/plasma.
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