Site Directed Mutagenesis (SDM) Mouse Point mutation 3T3-L1

Get tips on using Cell Proliferation ELISA, BrdU to perform Cell cytotoxicity / Proliferation assay cell type - 3T3-L1

Get tips on using CM-H2DCFDA (General Oxidative Stress Indicator) to perform ROS assay cell type - 3T3-L1

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

Get tips on using Xfect™ Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines 3T3-L1

Get tips on using GeneJuice® Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines 3T3-L1

Get tips on using MTT Cell Viability Assay Kit to perform Cell cytotoxicity / Proliferation assay cell type - 3T3-L1

Get tips on using Calcein AM Cell Viability Assay Kit to perform Live / Dead assay mammalian cells - 3T3-L1

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.

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines 3T3-L1