0.5 × 106 DCs/tube were spun down (300 g, 5 min), washed once, and then resuspended in FACS buffer (1× PBS, 5 mM EDTA, 1% BSA). Cells were first stained with Fc-block (1:100; 93) and then with a mixture of anti-mouse Cd11c-APC (1:300; N418) and anti-mouse MHC II–eFluor450 (1:800; M5/114.15.2; all eBioscience). All stainings were performed at 4°C for 15 min. Cells were washed once with 1 ml FACS buffer, resuspended, and analyzed on a FACS Canto II machine (BD Biosciences).
M5 114
Manufactured by Thermo Fisher Scientific
Sourced in United States
The M5/114.15.2 is a laboratory instrument used for cell sorting and analysis. It is designed to separate and identify specific cell types within a sample based on their physical and biochemical properties. The device utilizes flow cytometry technology to perform these functions.
Automatically generated - may contain errors
Lab products found in correlation
M1 70, by Thermo Fisher Scientific (4 mentions)
Lsrfortessa, by BD (3 mentions)
Il 22 poly5164, by BioLegend (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Fc block 2.4g2, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Live dead dye, by Thermo Fisher Scientific (2 mentions)
Cd16 32, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Ds5mmer, by Thermo Fisher Scientific (2 mentions)
Afs98, by BioLegend (2 mentions)
Facsaria, by BD (2 mentions)
Fc block, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd11c apc, by Thermo Fisher Scientific (1 mentions)
Facs canto 2 machine, by BD (1 mentions)
Background blocker, by Diagnostic BioSystems (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Chaps pbs, by AppliChem (1 mentions)
Ziptip c18 pipette tips, by Merck Group (1 mentions)
Sepharose, by GE Healthcare (1 mentions)
19 protocols using m5 114
1
Dendritic Cell Phenotyping Protocol
2
Epidermal Sheet Immunofluorescence and Immunohistochemistry
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For the IF procedure, epidermal sheets were rinsed in 1x PBS that was supplemented with 0.3% Triton X-100 and blocked for 2 hours at 4°C with background blocker (Diagnostic Biosystems, CA, USA); the epidermal sheets were then washed three times with 1x PBS and incubated overnight at 4°C with an anti-CD207 antibody (donated by Juliana Idoyaga, Ph.D., Rockefeller University). Next, the sheets were incubated with a Texas Red-labeled secondary antibody (Vector, Burlingame, CA, USA) for 2 hours at room temperature. Finally, the sheets were rinsed as described above, counterstained with DAPI, and mounted in Vectashield (Vector, Burlingame, CA, USA).
For IHC, the epidermal sheets were incubated with PolyDetector Peroxidase Block quenching buffer (Bio SB, CA, USA) and 0.1% BSA and then incubated with a primary antibody against MHC-II (1 : 50) (M5/114.15.2; eBioscience, CA, USA) or an anti-CD205 antibody (1 : 25) (NLDC-145; AbD Serotec, NC, USA). After the sheets were washed in 1x PBS, they were incubated with a secondary antibody (1 : 200) (anti-rat HRP; eBioscience, CA, USA). Proteins were detected using the DAB chromogen (0.3 mg/mL DAB; Bio SB, CA, USA). Sheets that had not been incubated with primary antibody were included as negative controls.
For IHC, the epidermal sheets were incubated with PolyDetector Peroxidase Block quenching buffer (Bio SB, CA, USA) and 0.1% BSA and then incubated with a primary antibody against MHC-II (1 : 50) (M5/114.15.2; eBioscience, CA, USA) or an anti-CD205 antibody (1 : 25) (NLDC-145; AbD Serotec, NC, USA). After the sheets were washed in 1x PBS, they were incubated with a secondary antibody (1 : 200) (anti-rat HRP; eBioscience, CA, USA). Proteins were detected using the DAB chromogen (0.3 mg/mL DAB; Bio SB, CA, USA). Sheets that had not been incubated with primary antibody were included as negative controls.
3
Isolation of MHC Class II Ligands
4
Flow Cytometry-based Epidermal ILC Isolation
5
Flow Cytometry Analysis of Macrophage Phenotype
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To evaluate cell viability, cells were detached from plates using fresh EDTA (5 mM) in PBS, pooled with cell supernatants containing propidium iodide (PI, 10 μg/mL) and recorded using an LSR II flow cytometer (Becton Dickinson, NJ). PI exclusion analysis for each sample was performed with 10,000 single cell, non-debris events using a FSC-A versus PI FACS plot. For cell surface marker expression analysis, stimulated BMDMs were harvested as described above and incubated with the following antibodies: F4/80-FITC (1:200, eBioscience; clone BM8), MHCII-AF700 (1:200, eBiocience; M5/114.15.2), CD206-APC (1:200, eBioscience; MR6F3) and CD11b-PE-Cy7 (1:400, eBioscience; M1/70) for 30 min on ice. Cells were washed once in PBS prior to analysis by flow cytometry. Analysis of cell surface marker expression was performed on 10,000 single cell, PI negative, gated events. Flow cytometry data were analyzed using WEASEL version 2.7 software (Frank Battye).
6
Isolation and Characterization of Immune Cells
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Human BLS cell lines and in vitro generated B cell mutants have been described and are established human cell lines [1 (link),30 (link)]. T cells were enriched using anti-CD4 and/or anti-CD8 magnetic beads (Miltenyi Biotec). For all flow cytometric analyses, gating on living cells and exclusion of doublets was performed. Enriched TEC suspensions [2 (link)] were washed in PBS, 2% FCS, 5mM EDTA and stained for flow cytometry using death exclusion markers (either DAPI or 7AAD), UEA1 (Sigma) and the following mAb-conjugated mix: α-CD45 (30F11, BioLegend) and α-BP1 (6C3; BioLegend), α-MHCII (M5/114.15.2; eBioscience) and α-EpCAM (G8.8, Developmental Studies Hybridoma Bank, Iowa), α-H2-Db (B22/24g) and α-H2-Kb (B8.24.3). Splenocytes were preincubated with anti-CD16/32 (2.4G2) to block FcRs and stained using Abs against CD8a (Ly-2), CD3e (145-2C11), CD4 (L3T4), CD11b (M1/70), CD11c (N418), CD19 (1D3), H2-Db (28-14-8), H2-Kb (AF6–88.5.5.3), MHCII (M5/114.15.2), NK1.1 (PK136), B220 (RA3-6B2), Qa-2 (69H1-9-9) (all from eBioscience). Streptavidin conjugated to different fluorophores was from eBioscience. Stainings were performed with appropriate combinations of fluorophores. Data was acquired with a FACSCanto flow cytometer (Becton Dickinson) and analyzed using FlowJo software (Tree Star).
7
Comprehensive Immune Cell Profiling
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Cells were stained with fixable LIVE/DEAD dye (Life Technologies), blocked with anti-CD16/32, and stained with antibodies for CD45 (30-F11; BioLegend), CD3 (17A2; BioLegend), CD4 (GK1.2; eBiosciences), CD11b (M1/70; BioLegend), CD11c (N418), CXCR3 (CXCR3–173), Ly6C (HK1.4; BioLegend), CD44 (IM7; BioLegend), CD62L (MEL-14; Invitrogen), and/or major histocompatibility complex class II (M5/114.15.2; eBiosciences). For IFN-γ production, the cells were stimulated for 6 hours with phorbol myristate acetate and ionomycin in the presence of brefeldin A and monensin for the final 4 hours. The cells were stained for extracellular markers and then fixed with fixation/permeabilization buffer, and intracellular staining for Foxp3 and IFN-γ was performed in permeabilization buffer. Fluorescence minus one controls, single color controls, and an unstained control were used for all experiments. Data were collected on an LSRFortessa (BD) and were analyzed using FlowJo (BD). Microglia are defined as CD45intCD11b+, while inflammatory monocytes are CD45hiCD11b+Ly6c+.
8
Multiparametric Flow Cytometry Analysis
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Cell suspensions were incubated with Ghost Dye BV510 Live/Dead stain (Tonbo Biosciences, San Diego, CA) at room temperature for 20 min, washed, incubated with 1:250 Fc block (2.4G2, BD Biosciences, Franklin Lakes, NJ) at 4 °C for 10 min, and then incubated with fluorochrome-labeled antibodies at 4C for 30 min using the following antibodies: CD19-PerCP/Cyanine5.5 (1:400; 6D5, Biolegend), CD64-PE/Cy7 (1:200; X54-5/7.1, Biolegend), Ly6G-PE (1:200; 1A8, Biolegend), CD11b-APC-780 (1:200; M1/70, eBioscience), CD45-AF700 (1:200; 30-F11, eBioscience), F4/80-APC (1:100; BM8, Biolegend), CD90.2-BV570 (1:100; 30-H12, Biolegend), Gr-1-BV711 (1:200; RB6-8C5, Biolegend) , CD11c-Bv650 (1:200; N418, Biolegend), Ly6c-Bv605 (1:100; HK1.4, Biolegend), MHC Class II-e450 (1:400; M5/114.15.2, eBioscience). Cells were analyzed using an LSRII flow cytometer (BD Biosciences) through the UCSF Liver Center Flow Cytometry Core Facility. Analysis was performed with FlowJo v.10.7.1 (Tree Star, Ashland, OR).
9
Multiparameter Flow Cytometry Analysis
10
Isolation and Characterization of Murine Myeloid Cells
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The external muscle layer was washed using Hank's balanced salt solution containing Mg2+ and Ca2+, followed by digestion in buffer containing 2% fetal calf serum and 1.5 mg/mL collagenase VIII for 20 minutes at 37 °C with agitation. Samples were passed through a 70‐μm filter and suspended in 40% isotonic Percoll (Sigma), underlaid with 80% isotonic Percoll, and centrifuged at 1000 g with no brake for 20 minutes. Cells located at the interface were collected. Leukocytes were treated with Fc Block (2.4G2; BD Pharmingen) in Ca2+ and Mg2+ free HBSS buffer containing 10% fetal calf serum for 15 minutes on ice. Cells were then stained with fluorescent monoclonal antibodies against CD45 (30‐F11; eBioscience), CD11b (M1/70; eBioscience), CD11c (n418; eBioscience), F4/80 (BM8; eBioscience), and major histocompatibility complex (MHC) II (M5/114.15.2; eBioscience) for 20 minutes on ice. Further antibody details can be found in Table S2 .
To determine cell viability and count, 7‐Aminoactinomycin D (7‐AAD; BioLegend) and 5000 counting beads (335925; BD) were added to the cells, respectively. Samples were processed using an LSR‐Fortessa X20 (BD Biosciences), and the data were analyzed using FlowJo 10.0.7 (Tree Star).
To determine cell viability and count, 7‐Aminoactinomycin D (7‐AAD; BioLegend) and 5000 counting beads (335925; BD) were added to the cells, respectively. Samples were processed using an LSR‐Fortessa X20 (BD Biosciences), and the data were analyzed using FlowJo 10.0.7 (Tree Star).
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