To quantify autophagosomes, corrected (SFTPCtdT/WT) or mutant (SFTPCI73T/tdT) iAEC2s were transduced with an LC3:GFP encoding lentivirus (Twig et al., 2008 (link)) at an MOI of 20 in CK+DCI media containing polybrene (5 mg/ml). iAEC2s were then replated in growth factor reduced 3D Matrigel (Corning) at a concentration of approximately 400 cells/μl in CK+DCI media for expansion. Single cell suspension of transduced iAEC2s was achieved by incubation with 2 mg/ml dispase (Thermo Fisher Scientific) for 30–60 minutes at 37°C and subsequent incubation with 0.05% trypsin for 12–15 minutes at 37°C, as previously described (Jacob et al., 2019 (link)). Transduced iAEC2s were plated onto Greiner CELLview glass-bottom 4-compartment dishes (Greiner Bio-One GmbH) pre-coated with growth factor reduced Matrigel (Corning) in CK+DCI media. After 3 days, iAEC2s were imaged at 37°C and 5% CO2, using a Zeiss LSM 710-Live Duo Scan confocal microscope (Carl Zeiss AG, Oberkochen) (488 nm excitation), and GFP+ punctate autophagosomes were quantified by visual inspection in 40–50 cells per group before and after 1 hour of incubation with 50 nM bafilomycin A1 (LC Laboratories). LC3 and p62 were further quantified by western blot as described below.
Lsm 710 live duo scan confocal microscope
Manufactured by Zeiss
The LSM 710-Live Duo is a high-performance confocal microscope designed for live-cell imaging. It features a dual-scanning system that allows for fast, simultaneous acquisition of multiple channels. The microscope is equipped with various laser options and a wide range of detector configurations to support a variety of fluorescent applications.
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Lab products found in correlation
Dispase, by Thermo Fisher Scientific (2 mentions)
Growth factor reduced matrigel, by Corning (2 mentions)
Growth factor reduced 3d matrigel, by Corning (2 mentions)
Cellview glass bottom 4 compartment dishes, by Greiner (2 mentions)
Bafilomycin a1, by LC Laboratories (2 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (2 mentions)
Triton x, by Merck Group (2 mentions)
Hoechst 3342, by Thermo Fisher Scientific (2 mentions)
Hnf4a, by Abcam (2 mentions)
Anti aat, by Santa Cruz Biotechnology (2 mentions)
Nucblue live readyprobe reagent, by Thermo Fisher Scientific (1 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (1 mentions)
P35gc 1.5 14 c, by MatTek (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
6 protocols using lsm 710 live duo scan confocal microscope
1
Quantifying Autophagosome Formation in iAEC2s
2
Quantifying Autophagosome Formation in iAEC2s
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To quantify autophagosomes, corrected (SFTPCtdT/WT) or mutant (SFTPCI73T/tdT) iAEC2s were transduced with an LC3:GFP encoding lentivirus (Twig et al., 2008 (link)) at an MOI of 20 in CK+DCI media containing polybrene (5 mg/ml). iAEC2s were then replated in growth factor reduced 3D Matrigel (Corning) at a concentration of approximately 400 cells/μl in CK+DCI media for expansion. Single cell suspension of transduced iAEC2s was achieved by incubation with 2 mg/ml dispase (Thermo Fisher Scientific) for 30–60 minutes at 37°C and subsequent incubation with 0.05% trypsin for 12–15 minutes at 37°C, as previously described (Jacob et al., 2019 (link)). Transduced iAEC2s were plated onto Greiner CELLview glass-bottom 4-compartment dishes (Greiner Bio-One GmbH) pre-coated with growth factor reduced Matrigel (Corning) in CK+DCI media. After 3 days, iAEC2s were imaged at 37°C and 5% CO2, using a Zeiss LSM 710-Live Duo Scan confocal microscope (Carl Zeiss AG, Oberkochen) (488 nm excitation), and GFP+ punctate autophagosomes were quantified by visual inspection in 40–50 cells per group before and after 1 hour of incubation with 50 nM bafilomycin A1 (LC Laboratories). LC3 and p62 were further quantified by western blot as described below.
3
Imaging Panc1 Cells with Fluorescent Antibody
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Panc1 were seeded onto 35 mm No. 1.5 Coverslip poly-D-lysine coated plates (Mattek, P35GC-1.5-14-C). Binding was performed at 4 °C with 10 μg/ml hu6g8AF568. The nucleus was labeled using NucBlue Live Ready Probe Reagent (ThermoFisher, R37605). Live Cell Imaging Solution (Invitrogen, A14291DJ) was used during image acquisition, and was pre-warmed to 37 °C to induce antibody internalization. Imaging was performed on an LSM 710-Live Duo Scan confocal microscope (Carl Zeiss, White Plaines, NY) with humidified (37 °C, 5% CO2) Pecon stage-top incubation system. Additional information is listed in Supplementary Methods (see Additional file 1 ).
4
Immunofluorescence Staining of BCG-Infected Macrophages
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BMDMs from C57BL/6J mice were grown on coverslips and infected with BCG:gfp followed by treatment with rocaglate for 24 h. Cells were fixed with chilled 100% methanol for 5 min at room temperature and then blocked for 60 min with 1% BSA containing 22.52 mg/mL glycine in PBST (PBS+ 0.1% Tween 20). Cells were incubated with primary antibodies purchased from cell signaling technology [p62(1:200) and LC3B(1:200)] overnight at 4 °C in 1% BSA, and incubated with Alexa Fluor 594-conjugated Goat anti-Rabbit IgG (H+L) secondary Antibody (Invitrogen) in 1% BSA in dark for 1 h. The cells were mounted using ProlongTM Gold antifade reagent (Thermo Fisher Scientific) and Images were acquired using Zeiss LSM 710-Live Duo scan confocal microscope. All images were processed using ImageJ software.
5
Hepatocyte Differentiation and Mitochondrial Imaging
6
Hepatocyte Differentiation and Mitochondrial Imaging
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For immunohistochemistry analysis, cells were passaged at day 5 of differentiation into 2-well chamber slides for the remainder of hepatic directed differentiation (ThermoFisher Scientific). For mitochondrial morphology analysis MitoTracker Deep Red FM (ThermoFisher Scientific) was applied at 200nM for 45 min prior to fixation. Cells were fixed using 4% paraformaldehyde for 10 min at room temperatures. For antibody labeling, cells were permeabilized with 0.3% Triton X-(MilliporeSigma) and blocked with 4% normal donkey or goat serum prior to incubation overnight with primary antibodies. Cells were probed using anti-AAT (Santa Cruz Biotechnologies), HNF4A (Abcam), or 2C1 (a kind gift from Elena Miranda and David Lomas) antibodies. Following incubation with primary antibody, cells were washed in PBS with 0.05% Tween20 (MilliporeSigma) and then incubated with secondary antibodies, anti-rabbit IgG-AlexaFluor647 and anti-mouse IgG-AlexaFluor488, for 1h at room temperature. Finally, cells were again washed with PBS containing 0.05% Tween20 and then counterstained with Hoechst 3342 (ThermoFisher Scientific). Cells were imaged using the Zeiss LSM 710-Live Duo scan confocal microscope and images were processed with ImageJ software.
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