The viability and apoptotic rate of Caco-2 cells were quantified using the Muse Annexin V and Dead Cell Assay (Merck), according to the manufacturer’s instructions. After trypsinization and collection, Caco-2 cells were washed 3 times with PBS, resuspended in PBS + 1% FBS (v/v) and 1 volume of Annexin V reagent, and incubated for 20 min at RT in the dark. The analysis was performed using a Muse Cell Analyzer (Merck). Autophagy detection was assayed using the Autophagy LC3 Antibody-based Kit (Merck). Specifically, Caco-2 cells were permeabilized and then incubated in ice for 30 min with the anti-LC3 mouse monoclonal AlexaFluor555 conjugated antibody, according to the manufacturer’s instructions. The analysis was performed using the Muse Cell Analyzer (Merck).
Autophagy lc3 antibody based kit
Manufactured by Merck Group
Sourced in United States
The Autophagy LC3-antibody-based kit is a laboratory equipment product designed for the detection and quantification of LC3, a key protein involved in the process of autophagy. The kit provides the necessary reagents and materials for researchers to perform experiments related to autophagy, a fundamental cellular process.
Automatically generated - may contain errors
5 protocols using autophagy lc3 antibody based kit
1
Quantification of Caco-2 Cell Viability and Autophagy
2
Quantifying Autophagy in Caco-2 Cells
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After trypsinization, Caco-2 cells were fixed using 4% of paraformaldehyde for 15 min at RT. Then, LC3-II labelling was performed using the Autophagy LC3 Antibody-based Kit (Merck) according to the manufacturer’s instructions in order to validate the results obtained using the Muse Cell Analyzer (Merck). Finally, 1 × 106 cells were resuspended in 200 µL of D-PBS for the analysis and 2000 events were collected for every sample. Spot-count analysis was performed using the spot-count feature Spot Count_Range(Peak(Spot(M03,Ch3-LC3-AF555,Bright,7,3),Ch3,Bright,0).
3
Quantifying Autophagic Vacuoles by Flow Cytometry
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For analyzing autophagic vacuoles, the cells were treated similar to previous experiments, and subsequently washed and stained using the Autophagy LC3-antibody-based kit (Millipore), according to the manufacturer's instructions. The cells were incubated with Autophagy Reagent A in Earle's balanced salt solution (EBSS) for 5 h at 37°C, followed by washing with ice cold HBSS. The cells were then stained with anti-LC3 Alexa Fluor® 555 in 1X Autophagy Reagent B on ice for 30 min in the dark, and washed with ice cold 1X Assay Buffer. The stained samples were quantified by flow cytometry using a Muse Cell Analyzer (Millipore) in duplicates. Based on manufacture's instruction, at least 1,000 events for each sample should be acquired to assure statistically significant determination of stained cells number. These quantified results were presented as autophagy induction ratio (test sample fluorescence relative to control) after analysis using the MuseSoft 1.4.0.0. software.
4
Quantification of Autophagy via LC3 Staining
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For the analysis of autophagic vacuoles, the cells after treatment were washed and stained using the Autophagy LC3-antibody-based kit (Millipore, Hayward, CA, USA) according to the manufacturer’s instructions. Briefly, the cells were incubated with Autophagy Reagent A in Earle’s balanced salt solution (EBSS) for 5 h at 37 °C to protect the autophagosome-associated LC3 (LC3-II) protein against degradation while washing away cytosolic LC3 (LC3-I). Then, the cells were washed with ice cold HBSS and stained with anti-LC3 Alexa Fluor®555 in 1× Autophagy Reagent B on ice for 30 min in the dark. Next, the excess of dye was washed out with ice cold 1× Assay Buffer and the samples were quantified by flow cytometry in Muse Cell Analyzer (Millipore, Hayward, CA, USA). Autophagy Induction Ratio (test sample fluorescence relative to control) was analyzed with MuseSoft 1.4.0.0.
5
Quantifying Autophagic Vacuoles by LC3-II Flow Cytometry
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LC3-II flow cytometry analysis was carried out in accordance with the manufacturer’s protocol. Following the treatment, the cells were washed and stained for autophagic vacuoles using the Autophagy LC3-antibody-based kit (Millipore). Briefly, after the indicated treatment, the cells were incubated with Autophagy Reagent A in EBSS for 5 h at 37 °C to prevent autophagosome-associated LC3 (LC3-II) from degradation while washing away cytosolic LC3 (LC3-I). Then, the cells were washed with ice-cold HBSS and stained with anti-LC3 Alexa Fluor®555 in 1× Autophagy Reagent B on ice for 30 min in the dark. Next, the excess of the dye was washed out with ice cold 1× Assay Buffer, and samples were quantified by Muse Cell Analyzer (Millipore). The assay allows for the determination of the Autophagy Induction Ratio (test sample fluorescence relative to control) with the software MuseSoft 1.4.0.0 (Millipore).
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