protein-expression-and-purification-mammalian-cells-hek-293-mt-pa

Get tips on using HeV NCORE pET43.1a to perform Protein Expression Prokaryotic cells - E. coli HeV N

Get tips on using CelLytic™ M to perform Protein isolation Mammalian cells - Caco-2

Get tips on using CelLytic™ M to perform Protein isolation Mammalian cells - SH-SY5Y

Get tips on using CelLytic™ M to perform Protein isolation Mammalian cells - BHK-21

Get tips on using pGETS118 to perform Protein Expression Prokaryotic cells - B. subtilis cellulosomal complexes

Get tips on using Live and Dead Cell Assay (Abcam) to perform Live / Dead assay mammalian cells - L29 mouse fibroblast

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include: 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi have been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining efficacy of transduction and shRNA on its target site.

Cells are sourced from various tissues to grow them in in-vitro conditions. Therefore, cell specific nutrients are important for their survival, maintenance and growth. Determining the appropriate cell culture media is a challenge if you are growing a cell line or a microorganism for the first time. Established cell lines, primary cells, stem cells, bacteria and Yeast all require varied nutrients from basic to complex. Based on the cell type, one can easy find what media and nutrients your peers have used before you try to reinvent the wheel.

Cells are sourced from various tissues to grow them in in-vitro conditions. Therefore, cell specific nutrients are important for their survival, maintenance and growth. Determining the appropriate cell culture media is a challenge if you are growing a cell line or a microorganism for the first time. Established cell lines, primary cells, stem cells, bacteria and Yeast all require varied nutrients from basic to complex. Based on the cell type, one can easy find what media and nutrients your peers have used before you try to reinvent the wheel.

Cell cytotoxicity assays measure the ability of certain compounds or chemical mediators to reduce the viability of the cells. The term cell cytotoxicity assay can sometimes be used interchangeably with cell proliferation assay. Healthy living cells can be identified by the use of formazan dyes, protease biomarkers or by measuring ATP content. The formazan dyes are chromogenic products formed by the reduction of tetrazolium salts by dehydrogenases, such as lactate dehydrogenase (LDH) and reductases that are released during cell death. Common tetrazolium salts include INT, MTT, MTS and XTT. Cell cytotoxicity can also be measured by using the SRB and WST-1 assays. These assays can usually be used in a high-throughput fashion and can be quantitated by measuring absorbance, colorimetry or luminescence. All these assays require similar numbers of cell plating at the initiation, a time course of treatment with the cytotoxic agent and at least triplicates for each condition at every point of analysis. Cell shrinkage, plasma membrane blebbing, cell detachment, externalization of phosphatidylserine, nuclear condensation and ultimately DNA fragmentation are well-described features of apoptosis. The assays that rely on cell membrane integrity for their function, may not be able to quantify early apoptosis. Therefore, in order to distinguish early apoptotic vs. late apoptotic or necrotic cells, additional flow cytometry techniques can be used. A combination of Annexin V and PI (propidium iodide) can be used to distinguish early (Annexin V+/PI-) and late apoptotic (Annexin V+/PI+) cells. Sometimes, caspase assays are used in order to differentiate the stages of apoptosis.