Human iPSCs or derived‐EBs were dissociated to into a single‐cell suspension by TrypLE (Life Technologies, Carlsbad, CA), and washed with a buffer for FACS (PBS plus 1% FBS and 1 mM EDTA). The cells were resuspended in the FACS buffer, and labeled with fluorochrome‐conjugated anti‐human CD34‐APC (Miltenyi Biotec, #130‐090‐954), CD45‐PE (Miltenyi Biotec, #130‐080‐201), CD45‐ Brilliant Violet 605 (BioLegend, San Diego, CA, #304042), CD235a‐Pacific Blue (BioLegend, #349108) antibodies. Isotype‐matched control antibodies were used to determine the background staining. The erythrocyte enucleation was evaluated by DRAQ5 staining (Thermo Scientific, #62251, 1:2,000 dilution). To characterize human iPSCs, the primary antibodies anti‐human TRA‐1‐60 (Millipore, Billerica, MA, #MAB4360) and SSEA‐4 (Millipore, #MAB4303) were used. Their recognitions were detected by respective secondary antibodies anti‐mouse IgM‐Alexa Fluor 555 (Thermo Scientific, #A21426) and anti‐mouse IgG‐Alexa Fluor 555 (Thermo Scientific, #A21422). Flow cytometric analysis was performed on a FACSCalibur or LSR II analyzer (BD Biosciences). Data analysis was performed using FlowJo or FCS Express software.
Cd45 brilliant violet 605
Manufactured by BioLegend
CD45-Brilliant Violet 605 is a fluorescently-conjugated antibody that binds to the CD45 antigen, a common leukocyte antigen expressed on the surface of various immune cells. It is designed for use in flow cytometry applications to identify and analyze different cell populations within a sample.
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Lab products found in correlation
Cd45 pe, by Miltenyi Biotec (1 mentions)
Ssea 4, by Merck Group (1 mentions)
Lsrii analyzer, by BD (1 mentions)
Anti mouse igg alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Anti human cd34 apc, by Miltenyi Biotec (1 mentions)
Anti human tra 1 60, by Merck Group (1 mentions)
Anti mouse igm alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Cd4 percp cy5, by BioLegend (1 mentions)
Live dead dye, by BioLegend (1 mentions)
Foxp3 fitc, by Thermo Fisher Scientific (1 mentions)
Ifn γ pe, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
2 protocols using cd45 brilliant violet 605
1
Multiparametric Flow Cytometry for Characterizing Human iPSCs and Derived Cell Types
2
Isolation and Characterization of Retinal and CDLN Cells
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Cells were isolated from the retina and CDLNs on day 14 after immunization. Dead cells were excluded using live/dead dye (#423105, BioLegend, San Diego, CA, USA). Then they were stained with the following antibodies: the surface markers included: CD4 Percp-Cy5.5 (#100434), CD45 Brilliant Violet 605 (#103155) (BioLegend), TGFBR2 PE (#FAB532P, R&D Systems). For intracellular cytokine staining, the cells were stimulated with 5 ng/mL of phorbol myristate acetate, 500 ng/mL ionomycin, and 1 mg/mL brefeldin A (Sigma) at 37 °C for 5 h, following by fixation and permeabilization for 30 min. Then, cells were stained with antibodies detecting: IFN-γ PE (#505808), IL-17A Alexa Fluor 647 (#506912), FOXP3 FITC (#11-5773-82), Id2 PE-Cy7 (#25-9475-82, Invitrogen). For the Pim1 staining, cells were stained with surface antibodies, fixed, permeabilized, stained with Pim1 antibody (#3247S), then stained with Alexa Fluor 488-labeled antibody (#4412S) (Cell Signaling Technology, Danvers, USA). Finally, the cells were kept overnight at 4 ◦C and measured by flow cytometry. The flow cytometer (BD LSRFortessa, USA) was used for analysis and the results were analyzed with FlowJo software (version 10.0.7, Tree Star, Ashland, OR, USA).
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