Site Directed Mutagenesis (SDM) Human Point mutation A549

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.

Cell Invasion or Cell Migration assays are technically challenging to set up as the gradient between the two compartments equilibrates in time during the assay. It is also problematic to view cells and for cells to migrate through a non-physiologic polycarbonate or polypropylene filter. Care must be taken while loading the well with cells to form a single cell suspension. Precaution must be taken while trypsinization (under-trypsinization can lead to cell clumping while over-trypsinization could strip off adhesion molecules necessary for migration). This leads to difficulty in getting significant results, when only small numbers of cells cross the filter or when the distribution and/or staining of the cells is uneven.

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

Get tips on using siGENOME Human DAB2 (1601) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A549 DAB2

Get tips on using ON-TARGETplus Human CDK9 (1025) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A549 CDK9

Get tips on using ON-TARGETplus Human IRF3 (3661) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A549 IRF3

Get tips on using ON-TARGETplus Human GAPDH (2597) siRNA - Individual to perform siRNA / miRNA gene silencing Human - A549 GAPDH

Get tips on using Accell Human VDAC1 (7416) siRNA - Set of 4 to perform siRNA / miRNA gene silencing Human - A549 VDAC1