Footpad and pLN cells were resuspended in blocking buffer (1% v/v rat and mouse serum) for 20 min. Live cells were stained with a Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen) for 30 min before staining with the following cell-specific markers following manufacturer’s protocol: APC-Cy7-conjugated rat anti- mouse CD45 (BD Biosciences), PE-Cy7–conjugated rat anti-mouse CD3 (Biolegend), Pacific Blue-conjugated rat anti-mouse CD4 (Biolegend), PE-conjugated rat anti-mouse CD11b (BD Biosciences) and APC-conjugated rat anti-mouse Ly6G (Biolegend) for 20 min at room temperature. Cells were subsequently washed and fixed with 100 µl neat IC fixation buffer (eBioscience) for 5 min. Cells were then washed and resuspended for flow cytometry data acquisition. Data were acquired BD FACSCanto II (BD Biosciences) with FACSDiva software. Analyses were performed using FlowJo (Version 10) (Tree Star).
Apc cy7 conjugated rat anti mouse cd45
Manufactured by BD
APC-Cy7-conjugated rat anti-mouse CD45 is a fluorochrome-conjugated antibody that binds to the CD45 protein, a common leukocyte antigen expressed on the surface of mouse immune cells. This product is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Apc conjugated anti cd206, by Thermo Fisher Scientific (2 mentions)
Pe conjugated rat anti mouse cd11b, by BD (1 mentions)
Apc conjugated rat anti mouse ly6g, by BioLegend (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Fitc conjugated cd3e, by BD (1 mentions)
Alexa fluor 546 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Biotinylated uea 1, by Vector Laboratories (1 mentions)
7 aminoactinomycin d, by Fujifilm (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Bv510 conjugated anti cd86, by BD (1 mentions)
Fitc conjugated rat anti cd11b, by BD (1 mentions)
Fixation permeabilization concentrate, by Thermo Fisher Scientific (1 mentions)
3 protocols using apc cy7 conjugated rat anti mouse cd45
1
Immune Cell Phenotyping by Flow Cytometry
2
Multiparametric Flow Cytometry Analysis of Immune Cell Populations
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Rabbit anti–mouse keratin-5 antibody was purchased from Covance. Biotinylated UEA-1 was obtained from Vector Laboratories. The APC-Cy7–conjugated rat anti–mouse CD45, PE-Cy7–conjugated TER119, and PE-conjugated anti–mouse EpCAM (G8.8) antibodies were obtained from BD or BioLegend. The PE-conjugated rat anti-CD45, PE-conjugated mouse TER-119, FITC-conjugated mouse MHC II (M5/114.15.1), and APC-conjugated Aire antibodies were obtained from eBioscience. PE-conjugated CD80, CD86, CD40, and PD-L1 antibodies, purified anti–mouse CD16/32, FITC-conjugated ant–mouse EpCAM (G8.8), APC-Cy7–conjugated CD4, PE–Cy-7–conjugated CD8, and APC-conjugated GITR antibodies were obtained from BioLegend. PE-conjugated CD8, FITC-conjugated CD4, and FITC-conjugated CD3e antibodies were obtained from BD. PE-conjugated anti-Foxp3 (FJK-16s) was purchased from eBioscience. 7-aminoactinomycin D and recombinant mouse RANKL were purchased from Wako Pure Chemical Industries. Anti–mouse OPG antibody was purchased from R&D Systems. A RANK-Fc chimera was obtained from Sigma-Aldrich. Alexa Fluor 546–conjugated streptavidin and Alexa Fluor 488–conjugated anti–rabbit IgG were obtained from Invitrogen.
3
Microglial Phenotyping by Flow Cytometry
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Flow cytometry was used to assess microglial markers. A Neural Tissue Dissociation Kit (MACS) was used to homogenize the brains of the mice according to the instructions (Miltenyi Biotec). A Percoll gradient was used to collect monocyte‐enriched cells.38 Cells were labeled with APC‐Cy7‐conjugated rat antimouse CD45 (BD, 557659), FITC‐conjugated rat anti‐CD11b (BD, 553310), PerCP‐Cyanine5.5‐conjugated F4/80 (Invitrogen, 45‐4801‐82), APC‐conjugated anti‐CD206 (Invitrogen, 17‐2061‐82) and BV510‐conjugated anti‐CD86 (BD, 563077) and suitable isotype controls according to the instructions (eBioscience). Before staining CD206, the cells were treated with Fixation/Permeabilization Concentrate (eBioscience, 00‐5123‐43, 00‐5223‐56) for 30 minutes and then incubated with APC‐conjugated anti‐CD206 in permeabilization buffer (eBioscience, 00‐8333‐56) for 30 minutes and analyzed on a FACSVerse cell sorter and studied with FlowJo software.
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