Phosphate-Buffered Saline (PBS, pH 7.4), penicillin–streptomycin, and ROS detecting probe were purchased from Keygen (Nanjing, China). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640, and trypsin were purchased from Thermo Fisher Scientific (MA, USA). Cell Counting Kit 8 (CCK-8), Annexin V-PI cell apoptosis kit, and JC-1 kit were purchased from Yeasen Biotechnology (Shanghai, China). Thiophene-2-thiol,4,6-dia-midino-2-10 phenylindole (DAPI), Mito-Tracker green, Golgi-Tracker green, ER-Tracker green, and Lyso-Tracker green were purchased from Beyotime (Shanghai, China). All fluorescent-labeled monoclonal antibodies were obtained from Biolegend (CA, USA) and Beyotime (Shanghai, China). αPD-1 antibody was supplied from BioXcell (NH, USA) and Zinc Phthalocyanine (ZnPc, 98%) was purchased from Adamas (Shanghai, China). All other chemical reagents were purchased commercially from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Fluorescent labeled monoclonal antibodies
Manufactured by BioLegend
Sourced in United States
Fluorescent-labeled monoclonal antibodies are laboratory reagents used in various immunological techniques. They consist of monoclonal antibodies that have been conjugated with fluorescent dyes. These labeled antibodies can be used to detect and quantify specific target molecules or cells in samples, such as in flow cytometry, immunofluorescence microscopy, and other applications that require the identification and analysis of cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Jc 1 kit, by Yeasen (1 mentions)
Golgi tracker green, by Beyotime (1 mentions)
αpd 1 antibody, by BioXCell (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Er tracker green, by Beyotime (1 mentions)
Penicillin streptomycin, by Keygen Biotech (1 mentions)
Cell counting kit 8 cck 8, by Yeasen (1 mentions)
Mito tracker green, by Beyotime (1 mentions)
Lysotracker green, by Beyotime (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Leukocyte activation cocktail, by BD (1 mentions)
Facsverse cytometer, by BD (1 mentions)
4 protocols using fluorescent labeled monoclonal antibodies
1
Comprehensive ROS-sensing Protocol
2
Monocyte CD64 Expression Analysis
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The expressions of CD64 on monocytes were determined with flow cytometry analysis with fluorescent-labeled monoclonal antibodies from BioLegend (San Diego, CA). Leukocytes stained with either FITC-conjugated anti-CD64 mAb (10.1, mIgG1) plus APC-conjugated anti-CD14 or mIgG1-FITC isotype control plus APC-conjugated anti-CD14 were analyzed on a FACS Canto flow cytometer (BD Biosciences). The FlowJo software (Tree Star Inc.) was used to evaluate flow cytometry data. Characteristic light-scatter properties were used to identify neutrophils and CD14+ monocytes cells were gated in flow cytometry for the geometric mean intensity of CD64 expression.
3
Isolation and Analysis of Adipose-Derived Immune Cells
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The adipose tissue stromal-vascular fraction was obtained as described previously (3 (link)) and resuspended in PBS supplemented with 2% FCS. Surface markers were stained with saturating amounts of the following fluorescent-labeled monoclonal antibodies (BioLegend) for flow cytometry: CD11c (clone N418), CD11b (clone M1/70), F4/80 (clone CI:A3–1), CD206 (clone C068C2), and CD45 (BD Horizon clone 30-F11). For intracellular cytokine staining, cells were stimulated in vitro for 5 h at 37°C in 5% CO2 with a leukocyte activation cocktail (BD Biosciences) and stained with anti–tumor necrosis factor (TNF) antibody (clone MP6-XT22; BioLegend) as described previously (3 (link)). Cells were acquired on a specially ordered five-laser LSR II Flow Cytometer (BD Biosciences) at the Beth Israel Deaconess Medical Center Flow Cytometry Core and analyzed with FlowJo software (Tree Star).
4
Immunophenotyping of Antigen-Presenting Cells
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After stimulation, cells were harvested by adding 2% EDTA+trypsin. Cells were washed twice with PBS-A. Membrane staining was performed using fluorescent-labeled monoclonal antibodies from BioLegend (San Diego, CA, USA), CD11c-FITC, HLA-DR-PeCy7, CD14-PercP, CD86-PE, ICOSL-APC, PDL1-BV421, and PDL2-APC, for 30 min at 4 °C.
After membrane staining, cells were fixed and permeabilized with D Cytofix/Cytoperm™ according to the manufacturer’s instructions. Then, cells were incubated with anti-IRF4-PE. Next, cells were washed twice with PBS-A, resuspended in 300 mL of PBS, and immediately acquired in a BD FACS Verse cytometer. Fluorescence Minus One (FMO) and the BD™ Anti-Mouse Ig, κ/Negative Control Compensation Particles Set (BD™ Comp Beads) were used as controls for fluorescence compensation.
After membrane staining, cells were fixed and permeabilized with D Cytofix/Cytoperm™ according to the manufacturer’s instructions. Then, cells were incubated with anti-IRF4-PE. Next, cells were washed twice with PBS-A, resuspended in 300 mL of PBS, and immediately acquired in a BD FACS Verse cytometer. Fluorescence Minus One (FMO) and the BD™ Anti-Mouse Ig, κ/Negative Control Compensation Particles Set (BD™ Comp Beads) were used as controls for fluorescence compensation.
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