biotinylated capture antibody  (Sino Biological)

Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet: Anticipated results. (a) Example images from a CRISPR knockout screen for regulators of NFkB activation in A549 cells43. Sequencing and phenotyping data (p65 localization) are shown for a single field of view. White outlines in sequencing images represent individual cells; colored outlines in the phenotype image represent clusters of neighboring cells with identical sgRNA assignments (scale bar, 50 μm). (b) Distribution of per-cell nuclear translocation scores after TNFɑ stimulation for non-targeting sgRNAs and sgRNAs targeting TNFRSF1A (TNFa receptor), MAP3K7 (downstream NFkB regulator), and IL1R1 (not involved in TNFa signaling). All sgRNAs were downsampled to a maximum of 100 cells to more easily compare distributions. (c) Kinetics of NFkB activation from a separate live-cell optical pooled screen performed in HeLa cells43. In unperturbed cells, p65 translocates from the cytoplasm to the nucleus ~45 min after stimulation, followed by a slower, partial relaxation back to the cytoplasm. Top, translocation traces for individual cells mapped to sgRNAs targeting TNFRSF1A, RIPK1, and TNFAIP3 (negative regulator of TNFa signaling). The gray curve indicates the average of cells assigned to non-targeting controls. Middle, per-sgRNA averages, with error bars indicating standard error of the mean (data downsampled to 300 cells per sgRNA). Bottom, per-gene distributions at fixed time points of all cells mapped to sgRNAs targeting the respective gene (green) or non-targeting sgRNAs (gray). The single-cell resolution of optical pooled screens reveals distribution features of perturbation effects, including bimodality and distribution width.

Article Snippet: Recombinant human IL-1β (Invivogen, cat. code rcyec-hil1b). . Fixed-cell Phenotyping Rabbit anti-p65 antibody (Cell Signaling Technology, cat. no. 8242; https://scicrunch.org/resolver/AB_10859369).

Techniques: CRISPR, Knock-Out, Activation Assay, Sequencing, Translocation Assay

Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet: Troubleshooting table

Article Snippet: Recombinant human IL-1β (Invivogen, cat. code rcyec-hil1b). . Fixed-cell Phenotyping Rabbit anti-p65 antibody (Cell Signaling Technology, cat. no. 8242; https://scicrunch.org/resolver/AB_10859369).

Techniques: Transformation Assay, Electroporation, Plasmid Preparation, Amplification, Transfection, Transduction, Fluorescence, Imaging, Flow Cytometry, Expressing, Transferring, In Situ, Sequencing, Evaporation, Incubation, Buffer Exchange, Staining, Diffusion-based Assay, Selection, Infection, Concentration Assay, Microscopy, Software

Journal: Acta Naturae

Article Title: A Highly Productive CHO Cell Line Secreting Human Blood Clotting Factor IX

doi:

Figure Lengend Snippet: The integrity of the intracellular and extracellular FIX polypeptide chain for the p1.1-F9-T2/S cell line, FIX secretion level and the change in clotting activity for various producer cell lines; the phylogenetic tree for the Chinese hamster VKORC1 protein. Panel A – Western blotting of the secreted and intracellular FIX. SDS-PAGE under reducing conditions, detection by polyclonal anti-FIX antibodies, the molecular weight of the marker bands is shown in kDa. Denotation: “K + ” – recombinant FIX standard; “control-” – untransfected CHO DG44 cells. The mature FIX position is depicted by an arrow. Panel B – FIX secretion level and the degree of propeptide processing for cell populations and clonal lines. ELISA-determined specific productivity is shown as bars (left axis). Specific productivity as the clotting activity is shown as a broken line (right axis). The percentage of FIX molecules without the propeptide was determined by ELISA and is shown as numbers above the bars. Specific productivity for both methods is shown as the mean value; error bars represent the standard deviation, n=2. The scheme of production of cell populations and clonal lines is shown with arrows. Panel C – The taxonomic tree for a mammalian VKORC1 protein visualized using the Tree Viewer software (NCBI, USA). The scale bar represents the evolutionary distance measured as the number of substitutions per amino acid residue. Panel D – multiple alignment of the amino acid sequences of VKORC1 variants for the selected mammalian species. Conservative amino acid residues are shown against a black background

Article Snippet: ELISA measurement of FIX concentration The FIX concentration was measured using rabbit anti-FIX polyclonal antibodies (LifeSpan BioSciences, USA) (50 ng/well) as an immobilized antibody according to the procedure described in [ ].

Techniques: Coagulation, Activity Assay, Western Blot, SDS Page, Molecular Weight, Marker, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation, Software, Residue