siRNA / miRNA gene silencing Human HeLa EPAS-1

Reporter gene assays enable high sensitivity measurement of gene expression and cell signaling through the addition of bioluminescent genes into target cells. One of the major challenges is to make a specific construct that has no responses other than those related to the signaling pathway of interest. This can be achieved by selecting highly specific reporter constructs containing only defined responsive elements and a minimal promoter linked to reporter enzymes such as luciferase

Reporter gene assays enable high sensitivity measurement of gene expression and cell signaling through the addition of bioluminescent genes into target cells. One of the major challenges is to make a specific construct that has no responses other than those related to the signaling pathway of interest. This can be achieved by selecting highly specific reporter constructs containing only defined responsive elements and a minimal promoter linked to reporter enzymes such as luciferase

Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).

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 GenElute™ Mammalian Total RNA Miniprep Kit to perform RNA isolation / purification Cells - immortalized HeLa

Get tips on using Mouse Sca-1/Ly6 APC-conjugated Antibody to perform Flow cytometry Anti-bodies Mouse - Ly-6A-E/Sca1

Get tips on using IL-4Rα shRNA (m) Lentiviral Particles to perform shRNA gene silencing Mouse - R221a IL4Rα

Get tips on using Mouse/Rat/Porcine/Canine TGF-beta 1 Quantikine ELISA Kit to perform ELISA Mouse - TGF-beta 1

Get tips on using Mouse/Rat/Porcine/Canine TGF-beta 1 Quantikine ELISA Kit to perform ELISA Rat - TGF-beta 1

Get tips on using JetPrime to perform DNA transfection Mammalian cells - Immortalized cell lines HeLa