Flowcytometry CD14

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.

Get tips on using Imprint® Chromatin Immunoprecipitation Kit to perform ChIP Mouse - CD4+ T

Get tips on using EpiTect Bisulfite Kit to perform DNA methylation profiling Whole genome profiling - mouse T-cell (CD4 / CD8)

Get tips on using Gentra Puregene Blood Kit to perform DNA isolation / purification Cells - Primary cells CD4+ T cells

Get tips on using EZ DNA Methylation kit to perform DNA methylation profiling Whole genome profiling - mouse T-cell (CD4 / CD8)

Get tips on using STEMdiff™ Hematopoietic Kit to perform Stem cell Differentiation media hiPSCs differentiation into CD43+ primitive hematopoietic progenitor cells (HPCs)

Get tips on using Nucleofector™ Kits for Human T Cells to perform DNA transfection Mammalian cells - Primary cells CD4+ T cells

Get tips on using PE Annexin V Apoptosis Detection Kit I to perform Apoptosis assay cell type - T-cells Mouse (CD4+ and CD8+)

Get tips on using FITC Annexin V Apoptosis Detection Kit I (RUO) to perform Apoptosis assay cell type - T-cells Mouse (CD4+ and CD8+)

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).