![]() ![]() For more information regarding TotalSeq™ antibody concentrations, please reach out to BioLegend Tech Services. Prepare antibody pool using titrated amounts (up to 1 µg) of each TotalSeq™, Cell Hashing, and/or biotinylated antibody.While cells are incubating in Fc Block, proceed to step 5.The final blocking volume should be 50 µL. Add 5 µL of Human TruStain FcX™ Fc Blocking reagent or 0.5 µL of TruStain FcX™ PLUS (anti-mouse CD16/32) antibody.If working with mouse cells, dilute 1 million cells in 49.5 μL of Cell Staining Buffer in 12 x 75mm flow cytometry tubes.If working with human cells, dilute 1 million cells in 45 μL of Cell Staining Buffer in 12 x 75mm flow cytometry tubes.Dilute cells in an appropriate volume prior to staining.For more information about the protocol used by BioLegend, see the following link, details can be found under “ PBMC viability assessment-general methods”. BioLegend uses a Countess II for counting and assessing cell viability, however other methods for assessing cell viability are suitable. Contact Technical Services with any questions regarding cell viability.If low cell viability is observed, users may need to enrich live cells or repeat cell suspension preparation. Low cell viability is associated with generation of poor data and is not ideal for single cell experimentation.Carefully count all cells to ensure accurate quantitation and assess cell viability. Optimization of staining conditions and concentrations may be required. Enzymatic digestion may result in cleavage of epitopes and result in reduced staining with TotalSeq™ antibodies. BioLegend has not tested this protocol using single cell suspensions derived from enzymatically digested tissue.If using cells isolated from whole lysed blood or other sample types, users may need to optimize staining concentrations. This protocol has been optimized using fresh human PBMCs isolated using Ficoll gradients and mouse splenocytes prepared using mechanical dissociation.DO NOT combine TotalSeq™-B and TotalSeq™-C antibodies in a single experiment. NOTE: The TotalSeq™ antibodies used will vary based on the nature of the experiment. (For use only with the Single Cell V(D)J Feature Barcoding kit)Ī biotinylated antibody and TotalSeq™-B barcoded streptavidinĪ biotinylated antibody and TotalSeq™-C barcoded streptavidinĬhromium Single Cell 3′ Feature Barcode Library Kit 16 rxns 1000079Ĭhromium Single Cell 5’ Library Construction Kit 16 rxns 1000020ĭual Index Kit NT Set A (for Feature Barcode Libraries) 96 rxns 1000242ĭual Index Kit TN Set A (for Feature Barcode Libraries) 96 rxns 1000250 (For use only with the Single Cell 3’ v3.1 Feature Barcoding kit) ![]() TotalSeq™-C antibodies and/or hashtag reagents TotalSeq™-B antibodies and/or hashtag reagents 12 x 75mm Falcon™ Round-Bottom Polystyrene Tubes (Fisher Scientific, Cat# 14-959-1A or equivalent).Flowmi™ Cell Strainer (Bel-Art, H-B Instrument, Cat# H13680-0040).Human TruStain FcX (Fc Receptor Blocking Solution, Cat.Please read the entire protocol before starting the experiments. The following protocol describes surface protein staining with TotalSeq™–B and TotalSeq™–C antibodies and/or hashtag antibodies, to enable protein detection in addition to Single Cell 3’ v3.1 and Single Cell V(D)J Feature Barcoding technology from 10x Genomics. For example, for any technology platform Buyer uses with TotalSeq™, it is Buyer's sole responsibility to determine whether it has all necessary third party intellectual property rights to use that platform and TotalSeq™ with that platform. TotalSeq™-B or -C with 10x Feature Barcoding Technologyīuyer is solely responsible for determining whether Buyer has all intellectual property rights that are necessary for Buyer's intended uses of the BioLegend TotalSeq™ products. ![]()
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