After generating a single cell suspension and staining for cpLECs with Ghost UV450 (Tonbo Biosciences, Catalog #: 13-0868-T500), CD31 Alexa 647 (BD Biosciences, Catalog #: 565608), Podoplanin PE (eBioscience, Catalog #: 12-5381-82), and CD45 APC-Cy7 (BioLegend, Catalog #: 103116), the cells were sorted with a FACS Aria III with a nozzle size of 130 μm at the UW Flow Core satellite facility in the UW Biotechnology Center. LECs were gated for singlets using both FSC and SSC, dead cells excluded by gating for Ghostnegative live cells, and LECs were identified using both CD31+ Podoplanin+ after excluding both CD45intermediate microglia and CD45+ leukocytes as previously described2 (link),6 (link),8 (link),19 . Cells were sorted into either FACS buffer (1% BSA in PBS) for scRNA-seq, or RPMI containing 10% FBS, 2-mercaptoethanol, and penicillin/streptomycin for in vitro co-culture experiments.
Ghost uv450
Manufactured by Cytek Biosciences
The Ghost UV450 is a fluorescent detection channel in Cytek Biosciences' flow cytometry instruments. It is designed to detect fluorescent signals in the ultraviolet wavelength range.
Automatically generated - may contain errors
Lab products found in correlation
Cd31 alexa 647, by BD (2 mentions)
Podoplanin pe, by Thermo Fisher Scientific (2 mentions)
Facs aria 3, by BioLegend (2 mentions)
Cd45 apc cy7, by BioLegend (2 mentions)
Cd11b m1 70, by Thermo Fisher Scientific (1 mentions)
Cytometric bead array, by BD (1 mentions)
Inos cxnft, by Thermo Fisher Scientific (1 mentions)
F4 80 bm8, by Thermo Fisher Scientific (1 mentions)
Ghost red, by Cytek Biosciences (1 mentions)
Facsdiva software, by BD (1 mentions)
B220 bv510, by BioLegend (1 mentions)
Pd l1 pe cy7, by BioLegend (1 mentions)
Cd4 buv496, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
4 protocols using ghost uv450
1
Isolation and Characterization of Lymphatic Endothelial Cells
2
Macrophage Phenotypic Analysis after LPS Stimulation
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After LPS stimulation for 0, 4 or 24 h as described above, macrophages were harvested after removal of supernatant and washing with PBS. For flow cytometry, cells were stained with viability dye (Ghost UV450 or Ghost Red710 Tonbo Bioscience, San Diego) using an intracellular staining protocol (IC fixation and permeabilization staining kit, eBioscience). Antibodies included iNOS (CXNFT, eBioscience catalogue no. 12-5920), CD11b (M1/70, eBioscience catalogue no. 25-0112), F4/80 (BM8, eBioscience catalogue no. 48-4801), Arginase-1 (polyclonal, R&D Systems catalogue no. IC5868F). All antibodies were used as 1:200 dilution. Macrophages were analysed by flow cytometry on an LSRII instrument using FACS Diva software (BD Bioscience) and analysed using FlowJo software (Tree Star, San Carlos, CA). The cytometric bead array (BD Bioscience) was used to determine cytokine concentrations in macrophage supernatant after LPS treatment according to manufacturers’ instructions.
3
Single-cell RNA-seq of cpLECs
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After generating a single cell suspension and staining for cpLECs with Ghost UV450 (Tonbo Biosciences, Catalog #: 13-0868-T500), CD31 Alexa 647 (BD Biosciences, Catalog #: 565608), Podoplanin PE (eBioscience, Catalog #: 12-5381-82), and CD45 APC-Cy7 (Biolegend, Catalog #: 103116) as previously described, the cells were sorted with a FACS Aria III with a nozzle size of 130 um at the UW Flow Core satellite facility in the UW Biotechnology Center. Cells were sorted into FACS buffer (1% BSA in PBS). A total of 25,102 cells from the control group and 53,224 cells from the experimental group post-sort were provided to the Biotechnology Core for scRNAseq.
4
Multiparameter Flow Cytometry Profiling
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After resuspension of mechanically dissociated cells in fluorescence-activated cell sorting (FACS) buffer (pH = 7.4, 0.1M PBS, 1 mM EDTA, 1% BSA), the cells underwent staining with conjugated antibodies/dyes. Conjugated antibodies were diluted 1:200 in FACS buffer, and the following antibodies and dyes were used: Ghost UV450 (Tonbo Biosciences, Catalog #: 13-0868-T500) to visualize live/dead cells; The copyright holder for this preprint this version posted October 9, 2020. ; https://doi.org/10.1101/2020.10.08.331801 doi: bioRxiv preprint (Biolegend, Catalog #: 100721); CD4 BUV496 to visualize T helper cells (BD Bioscience, Catalog #: 564667); B220 BV510 to visualize B cells (Biolegend, Catalog #: 103247); and PDL-1 PE-Cy7 to visualize the tolerogenic ligand PDL-1 (Biolegend, Catalog #: 124313). After staining for 30 minutes at 4ºC, cells were washed three times in FACS buffer and processed using a BD LSR II. For the CD45 IV timing experiments, cells were processed with Cytek's 3-laser Northern Lights.
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