Tumors were disaggregated in PBS/2% FBS and then incubated with PBS containing 2% FBS, 100 μg/mL collagenase A (Sigma, #10103586001), and 100 μg/mL DNase (STEMCELL, #07900) for 1.5 h at 37°C. Digested cell suspensions were passed through a 70-μm strainer. After centrifugation, cells were resuspended in PBS/2% FBS and, counted. 5×106 cells were labeled in 100 μL PBS containing 2% FBS, 2 μL FC blocker (BD Bioscience, #564220 for human and #553142 for mouse) and 0.4 μL eBioscience™ Fixable Viability Dye eFluor™ 506 (ThermoFisher Scientific, #65-0866-14) for 10 min at 4°C. Next, the following antibodies were next added: Pacific Blue™ anti-human CD45 (#304021), PerCP/Cyanine5.5 anti-human CD3 (#300328), Alexa Fluor® 700 anti-human CD4 (#357418), FITC anti-human CD8a (#300906), FITC anti-mouse CD8a (#100706), PerCP/Cyanine5.5 anti-mouse CD45.2 (#109828), Brilliant Violet 605™ anti-mouse CD4 (#100548), all from BioLegend; And BV786 Hamster Anti-Mouse CD3e, from BD Biosciences (#564379). Thirty min after incubation, cells were resuspended with PBS/2% FBS and analyzed by flow cytometry.
Pacific blue anti human cd45
Manufactured by BioLegend
Pacific Blue anti-human CD45 is a fluorochrome-conjugated antibody used to detect and quantify CD45-expressing cells in flow cytometry applications. CD45 is a receptor-linked protein tyrosine phosphatase that is expressed on the surface of all hematopoietic cells.
Automatically generated - may contain errors
Lab products found in correlation
Dnase, by STEMCELL (1 mentions)
Fc blocker, by BD (1 mentions)
Ebioscience fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions)
Percp cyanine5.5 anti human cd3, by BioLegend (1 mentions)
Fitc anti human cd8a, by BioLegend (1 mentions)
Collagenase a, by Merck Group (1 mentions)
Fitc anti mouse cd8a, by BioLegend (1 mentions)
Human il 10 elisa kit, by Cusabio (1 mentions)
Human il 1β elisa kit, by Cusabio (1 mentions)
Human tnf α elisa kit, by Cusabio (1 mentions)
Pe anti human cd274, by BioLegend (1 mentions)
3 protocols using pacific blue anti human cd45
1
Immune Cell Profiling from Tumor Samples
2
Macrophage Coculture with Glioma Stem Cells
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THP1-derived M1 macrophages were treated as follows: (i) 1 × 106 macrophages cultured alone; vi) 1 × 106 macrophages cocultured with 2 × 106 NSCs for 24 hours; (iii) 1 × 106 macrophages cocultured with 2 × 106 GSCs for 24 hours; (iv) 1 × 106 macrophages noncontact cocultured with 2 × 106 GSCs for 24 hours by 0.4 μmol/L Transwell, with GSCs placed in the upper chamber and BMDMs placed in the lower chamber; (v) 1 × 106 macrophages pretreated with 1 μmol/L cytochalasin D for 1 hour at 37°C and then cocultured with 2 × 106 U251 for 24 hours. ELISA and flow cytometry were performed on these cells.
1 × 106 THP1-derived M1 macrophages were cocultured with 2 × 106 GFP+ U251 for 24 hours, and then cells were stained with Pacific Blue anti-human CD45 (BioLegend, #982306, RRID:AB_2650649). CD45+GFP+ and CD45+GFP– cells were sorted by fluorescence-activated cell sorting (FACS) for ELISA and flow cytometry analysis.
The protein levels of IL1α, IL1β, TNFα, and IL10 in the macrophage culture supernatant were measured with Human IL1α ELISA Kit (Cusabio, #CSB-E04620h), Human IL1β ELISA Kit (Cusabio, #CSB-E08053h), Human TNFα ELISA Kit (Cusabio, #CSB-E04740h), and Human IL10 ELISA Kit (Cusabio, #CSB-E04593h), respectively. Experiments were conducted according to the manufacturer's instructions.
1 × 106 THP1-derived M1 macrophages were cocultured with 2 × 106 GFP+ U251 for 24 hours, and then cells were stained with Pacific Blue anti-human CD45 (BioLegend, #982306, RRID:AB_2650649). CD45+GFP+ and CD45+GFP– cells were sorted by fluorescence-activated cell sorting (FACS) for ELISA and flow cytometry analysis.
The protein levels of IL1α, IL1β, TNFα, and IL10 in the macrophage culture supernatant were measured with Human IL1α ELISA Kit (Cusabio, #CSB-E04620h), Human IL1β ELISA Kit (Cusabio, #CSB-E08053h), Human TNFα ELISA Kit (Cusabio, #CSB-E04740h), and Human IL10 ELISA Kit (Cusabio, #CSB-E04593h), respectively. Experiments were conducted according to the manufacturer's instructions.
3
Multiplex Immunophenotyping of Immune Cells
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Cells were stained with APC anti-human CD163 Antibody (BioLegend, #333610, RRID:AB_2074533), Alexa Fluor 647 anti-human CD206 (MMR) Antibody (BioLegend, #321116), PE anti-human CD274 (B7-H1, PD-L1) Antibody (BioLegend, #329706, RRID:AB_940368), APC anti-human CD273 (B7-DC, PD-L2) Antibody (BioLegend, #329708), PE anti-human CD276 (B7-H3) Antibody (BioLegend, #351004, RRID:AB_10720987), and Pacific Blue anti-human CD45 (BioLegend, #982306, RRID:AB_2650649) following the manufacturer's instructions.
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