To assess intracellular cytokines, leukocytes were stimulated for 4 hrs with phorbol 12-myristate 13-acetate (10 ng/ml), ionomycin (1 μg/ml) and brefeldin A (10 μg/ml). Cells were washed, fixed in 2% paraformaldehyde, permeabilized with 0.5% saponin buffer and stained with antibodies specific for CD4 (Biolegend – RM4-5), IFN-γ (eBioscience – XMG1.2), IL-4 (eBioscience – 11B11), IL-13 (eBioscience – eBio13A) and IL-17A (Biolegend – TC11-18H10.1). To assess cell populations and macrophage depletion, leukocytes isolated from lung tissue or 72 hrs thioglycollate elicited peritoneal cells (3% w/v) were labelled with a viability dye (Invitrogen – LIVE/DEAD Blue or Aqua), pre-incubated in 2% normal rat serum plus unlabelled anti-CD16/32 antibody (eBioscience – 93), and stained with combinations of antibodies specific for: CD11b (Biolegend – M1/70), CD11c (BD – HL3), CD45 (Biolegend - 30-F11), CD64 (Biolegend – X54-5/7.1), F4/80 (Biolegend – BM8), Gr-1 (Biolegend – RB6-8C5), Ly6C (Biolegend – HK1.4), Ly6G (BD – 1A8), MHC class II I-Ab (BD – AF6-120.1) or I-A/E (Biolegend M5/114.15.2), Siglec-F (BD – E50-2440), and TCRβ (eBioscience – H57-597). Flow cytometry was performed using a FACSCanto II or LSRII (BD Biosciences) and data were analyzed using FlowJo (Tree Star).
X54 5 7
Manufactured by BioLegend
The X54-5/7.1 is a multi-channel digital flow cytometer designed for advanced cell analysis. It features high-sensitivity detection, fast data acquisition, and a user-friendly interface. The core function of this instrument is to provide researchers with a powerful tool for comprehensive multiparameter analysis of complex biological samples.
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Lab products found in correlation
M1 70, by BD (3 mentions)
Rb6 8c5, by BioLegend (3 mentions)
M5 114, by BioLegend (3 mentions)
M1 70, by BioLegend (3 mentions)
Facscanto 2, by BD (2 mentions)
Rm4 5, by Thermo Fisher Scientific (2 mentions)
Ebio13a, by BioLegend (2 mentions)
Live dead blue or aqua, by Thermo Fisher Scientific (2 mentions)
Af6 120, by BioLegend (2 mentions)
E50 2440, by Thermo Fisher Scientific (2 mentions)
H57 597, by Thermo Fisher Scientific (2 mentions)
Tc11 18h10, by BioLegend (2 mentions)
Fjk 16, by Thermo Fisher Scientific (2 mentions)
E50 2440, by BD (2 mentions)
Fortessa x20, by BD (1 mentions)
Live dead near ir stain, by Thermo Fisher Scientific (1 mentions)
M1 69, by BioLegend (1 mentions)
Ra3 6b2, by BioLegend (1 mentions)
H57 597, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
8 protocols using x54 5 7
1
Multiparameter Flow Cytometry Analysis
2
Flow Cytometric Characterization of Murine Lung Cells
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Single-cell suspensions from lungs were prepared as described in the previous section. Cells were stained for a panel of murine cell surface markers and analyzed using a Fortessa X-20 (BD) and FlowJo software (Tree Star). Antibody clones used included RM4-5 (anti-CD4), 53–6.7 (anti-CD8α), and H57-597 (anti-TCRβ chain), all obtained from BD. In addition, H1.2F3 (anti-CD69), IM7 (anti-CD44), XMG-6 (anti-IFN-γ), N418 (anti-CD11c), M5/114.15.2 (anti-IA/IE), HK1.4 (anti-Ly6C), 1A8 (anti-Ly6G), M1/70 (anti-CD11b), 2E7 (anti-CD103), P84 (anti-CD172α), X54-5/7.1 (anti-CD64), BM8 (anti-F4/80), M1/69 (anti-CD24), RA3-6B2 (anti-B220), 4B12 (anti-CCR7), MP1-22E9 (anti-CSFR2α), AFS98 (anti-CD115), 323 (anti-CD40), and GL-1 (anti-CD86) were obtained from BioLegend. Clone 475301 (anti-CCR2) was obtained from R&D Systems. Live/Dead Near IR stain was obtained from Life Technologies and included in all staining protocols. Optimal antibody concentrations were determined in separate experiments, and appropriate fluorochrome-labeled isotype control antibodies used throughout. In all cases, cells were first gated on singlets (FSC-W vs. FSC-A or -H) and live cells (Live/Dead Near IR negative) before further analyses.
3
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).
4
Multiparameter Flow Cytometry Panel
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PD-1 FITC (1:300; J43; eBioscience), NK1.1 PE (1:300; PK136; eBioscience), CD3e PE-Dazzle594 (1:300; 145-2C11; BioLegend), CD3e PE (1:200; 145-2C11; eBioscience), FoxP3 PerCP-Cy5.5 (1:200; FJK-16s; eBioscience), CD8a PE-Cy7 (1:2000; 53-6.7; eBioscience), CD8a APC (1:600; 53-6.7; eBioscience), CD8a PerCP-Cy5.5 (1:400; 53-6.7; eBioscience), TCRb AF700 (1:100; H57-597; BioLegend), CD45 BV785 (1:1000; 30-F11; BioLegend), CD4 BV711 (1:400; GK1.5; BioLegend), CD19 BV650 (1:400; 6D5; BioLegend), CD19 PE (1:500; 1D3; eBioscience), CD11b BV605 (1:1000; M1/70; BioLegend), CD11b BV510 (1:400; M1/70; BioLegend), CD11c BV605 (1:400; N418; BioLegend), Siglec-F PE (1:300; E50-2440; BD Biosciences), F4/80 APC (1:200; BM8; BioLegend), Ly-6G AF 700 (1:300; 1A8; BioLegend), MHC II BV650 (1:4000; M5/114.15.2; BioLegend), CD64 BV421 (1:200; X54-5/7.1; BioLegend), TNF FITC (1:300; MP6-XT22; BioLegend), IFNg PE-Cy7 (1: 1000; XMG1.2; BD Biosciences), Eomes PE (1:200; W17001A; BioLegend), Ki-67 BV 650 (1:200, 11F6; Biolegend), GzmB FITC (1:100; GB11; BioLegend). Gating strategy is depicted in Supplementary Fig. 6d .
5
Multiparameter Flow Cytometry Analysis
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To assess intracellular cytokines, leukocytes were stimulated for 4 hrs with phorbol 12-myristate 13-acetate (10 ng/ml), ionomycin (1 μg/ml) and brefeldin A (10 μg/ml). Cells were washed, fixed in 2% paraformaldehyde, permeabilized with 0.5% saponin buffer and stained with antibodies specific for CD4 (Biolegend – RM4-5), IFN-γ (eBioscience – XMG1.2), IL-4 (eBioscience – 11B11), IL-13 (eBioscience – eBio13A) and IL-17A (Biolegend – TC11-18H10.1). To assess cell populations and macrophage depletion, leukocytes isolated from lung tissue or 72 hrs thioglycollate elicited peritoneal cells (3% w/v) were labelled with a viability dye (Invitrogen – LIVE/DEAD Blue or Aqua), pre-incubated in 2% normal rat serum plus unlabelled anti-CD16/32 antibody (eBioscience – 93), and stained with combinations of antibodies specific for: CD11b (Biolegend – M1/70), CD11c (BD – HL3), CD45 (Biolegend - 30-F11), CD64 (Biolegend – X54-5/7.1), F4/80 (Biolegend – BM8), Gr-1 (Biolegend – RB6-8C5), Ly6C (Biolegend – HK1.4), Ly6G (BD – 1A8), MHC class II I-Ab (BD – AF6-120.1) or I-A/E (Biolegend M5/114.15.2), Siglec-F (BD – E50-2440), and TCRβ (eBioscience – H57-597). Flow cytometry was performed using a FACSCanto II or LSRII (BD Biosciences) and data were analyzed using FlowJo (Tree Star).
6
Murine Bronchoalveolar Lavage and Cell Sorting
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Bronchoalveolar lavage was performed by first exposing the trachea of euthanized mice.
The exposed trachea was punctured using Vannas Micro Scissors (VWR), and 1 ml PBS was injected using a 20G‐1” IV catheter (McKesson) connected to a 1‐ml syringe.
The PBS was flushed into the lung and aspirated three times, and the recovered fluid was placed in a 15‐ml tube on ice.
The 1 ml PBS wash was then repeated three additional times for a total of 4 ml recovered fluid.
Cells were filtered, spun down, and resuspended in a 96‐well plate for antibody staining.
Cells were suspended in 1X PBS (pH 7.4) containing 0.01% NaN3 and 1% fetal bovine serum (i.e., FACS buffer).
7
Intestinal Immune Cell Profiling
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Isolated intestinal lamina propria cells were washed with FACS buffer (1× PBS containing 0.5% BSA and 2 mM EDTA), non-specific binding was blocked with FcR blocking antibody (anti-Mouse CD16/32, eBioscience, dilution 1:100) before staining with labeled monoclonal antibodies against CD45.2 (104, BD Pharmingen, dilution 1:160), MHCII (M5/114.15.2, BioLegend, dilution 1:160), CD11c (N418, eBioscience, dilution 1:160), CD11b (M1/70, BD Pharmingen, dilution 1:160), Ly6G (RB6-8C5, eBioscience, dilution 1:160), Ly6C (AL-21, BD Pharmingen, dilution 1:160), CD3 (145-2C11, BD Pharmingen, dilution 1:160), CD4 (RM4-5, BD Pharmingen, dilution 1:160), and CD64 (X54-5/7.1, BioLegend, dilution 1:160). Intracellular staining was carried out after permeabilization with Permfix (BD) for cytokines IL-17A (eBio17B7, eBioscience, dilution 1:100), IFN-γ (XMG1.2, eBioscience, dilution 1:100); or Transcription Factor Staining Buffer Set (eBioscience) for Foxp3 (FJK-16s, eBioscience, dilution 1:100)/IL-10 (JES5-16E3, eBioscience, dilution 1:100) staining. Dead cells were excluded with a fixable viability dye (eBioscience, dilution 1:500) before fixing. The fluorescence was examined on an LSRFortessaTM Flowcytometor (BD).
8
Detailed Tumor Immune Cell Analysis by FACS
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FACS analysis was performed using Guava® easyCyte 6HT or 8HT (Millipore Merck). For the cell-based-binding assay, FcγRI-transfected HEK293 cells (HEK293/FcγRI) were stained with BGB-A317 or BGB-A317/IgG4S228P or huIgG, followed by detection with AlexaFluor 488-conjugated goat F(ab′)2 anti-human IgG (F(ab′)2) fragment (Jackson ImmunoRes). The cell surface binding signals were quantified as mean fluorescence intensities (MFIs).
For the analysis of tumor infiltrated immune cells from the mouse in vivo cancer models, tumor tissue was cut into small pieces and digested with collagenase type I (1 mg/ml, Sigma) and 100 µg/ml DNase I (Sigma) in RPMI1640 plus 5% FBS for 30 min at 37 °C. Single cell suspension was obtained after passing the digested tissues through a 70 µm cell strainer. The cells were then washed and blocked by human IgG, followed by staining with human CD3 (HIT3a, 4A biotech), CD8 (OKT8, Sungene biotech), PD-1 (MIH4, eBioscience), CD64 (10.1, eBioscience), or mouse CD64 (X54-5/7.1, Biolegend) antibodies at 4 °C. The stained samples were subjected to flow cytometry and analysis using guavaSoft3.1.1 (Millipore, Merck).
For the analysis of tumor infiltrated immune cells from the mouse in vivo cancer models, tumor tissue was cut into small pieces and digested with collagenase type I (1 mg/ml, Sigma) and 100 µg/ml DNase I (Sigma) in RPMI1640 plus 5% FBS for 30 min at 37 °C. Single cell suspension was obtained after passing the digested tissues through a 70 µm cell strainer. The cells were then washed and blocked by human IgG, followed by staining with human CD3 (HIT3a, 4A biotech), CD8 (OKT8, Sungene biotech), PD-1 (MIH4, eBioscience), CD64 (10.1, eBioscience), or mouse CD64 (X54-5/7.1, Biolegend) antibodies at 4 °C. The stained samples were subjected to flow cytometry and analysis using guavaSoft3.1.1 (Millipore, Merck).
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