Autophagy assay cell type

Get tips on using 5 µm Chemotaxis Assays, 24-Well Format to perform Cell migration / Invasion cell type - A549

Get tips on using CytoTox 96® Non-Radioactive Cytotoxicity Assay to perform Live / Dead assay mammalian cells - CHO-K1

Get tips on using Qubit™ Protein Assay Kit to perform Protein quantification Mammalian cells - OUMS-27

Get tips on using QuantiPro™ BCA Assay Kit to perform Protein quantification Mammalian cells - HEK 293

Get tips on using Qubit RNA HS Assay Kit to perform RNA quantification Fuorimetric - human trophoblast cells

Get tips on using Qubit dsDNA HS Assay Kit to perform DNA quantification MCF10A breast epithelial cells

Reporter gene assays are designed to test the regulation of the expression of a gene of interest. This is usually done by linking the promoter of the gene of interest with a gene such as a firefly luciferase, which can be easily detected by addition of luciferin that leads to an enzymatic reaction to produce luminescence. The enzymatic reaction can be correlated to the expression of the gene of interest. Another luciferase gene that can be used is Renilla luciferase. For an appropriate luciferase assay: 1. the reporter should express uniformly in all cells, 2. specifically respond to effectors that the assay intends to monitor, 3. have low intrinsic stability to quickly reflect transcriptional dynamics. It is important to have an equal number of cells plated in each testing condition to avoid any incorrect readouts. Reporter assays could be single or dual reporter assays. The reporter could be both luciferases. Most dual-luciferase assays involve adding two reagents to each sample and measuring luminescence following each addition. Adding the first reagent activates the first luciferase reporter reaction; adding the second reagent extinguishes first luciferase reporter activity and initiates the second luciferase reaction. Dual-luciferase assays have some advantages, including 1. reduces variability, 2. reduces background, 3. normalizes differences in transfection efficiencies between samples.

An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

Get tips on using CytoTox 96® Non-Radioactive Cytotoxicity Assay to perform Live / Dead assay mammalian cells - mouse bone marrow-derived macrophages

Get tips on using Qubit RNA HS Assay Kit to perform RNA quantification Fuorimetric - human peripheral blood mononuclear cells (PBMCs)