siRNA / miRNA gene silencing Mouse 3T3-L1

Get tips on using Calcein AM Cell Viability Assay Kit to perform Live / Dead assay mammalian cells - 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 FlexiTube GeneSolution GS26354 for GNL3 to perform siRNA / miRNA gene silencing Human - MDA-MB-231 GNL3

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

Get tips on using PRO-PREP™ Protein Extraction Solution (C/T) to perform Protein isolation Mammalian cells - 3T3-L1

Get tips on using ROS-ID® Total ROS/Superoxide detection kit to perform ROS assay cell type - 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.

Get tips on using STEAP2 metalloreductase to perform siRNA / miRNA gene silencing Human - PC3 (human prostate cancer cell line) STEAP2

Get tips on using pcDNA™3.1 (+) Mammalian Expression Vector to perform siRNA / miRNA gene silencing Human - U251 cofilin-1 (CFL1)

Isolating DNA from tissues and paraffin-embedded tissue samples can be challenging as double-stranded DNA is physically fragile and highly susceptible to exo- and endonucleases. The best solution is to slice the tissues into smaller pieces and make a homogenate solution (using tissue homogenizer or grinding liquid nitrogen frozen samples) in the presence of DNAse inhibitors. Further, extracting DNA from the nucleus need specific methods by combining physical, mechanical and chemical lysis approaches,