LEC and HUVEC cells were washed with PBS and detached using Accutase (Innovative Cell Technologies, San Diego, CA, USA). Detached cells were incubated with anti-PE-conjugated human integrin αvβ3 antibody (Table S1 ) for 30 min at 4 °C. Cells were then washed with 0.5% BSA/0.1% sodium azide in PBS (FACS buffer). Ten thousand cells per sample were analyzed using a Novocyte flow cytometer (ACEA Biosciences, San Diego, CA, USA). Tumors were dissociated into single cells using mouse Tumor Dissociation Kits, and a gentleMACS™ Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Single cells were incubated with 10 μg/mL of anti-mouse CD16/32 antibody (clone 93, Biolegend, San Diego, CA, USA) in FACS buffer for 10 min at 4 °C to block Fc receptors. After washing the cells with FACS buffer, cells were stained with fluorophore-labeled antibodies (listed in Table S1 ) for 30 min at 4 °C. After washing, cells were stained with 7-AAD (5 μg/mL, Biolegend, #42040) for 5 min at 25 °C to determine cell viability. Cells were analyzed using a Novocyte flow cytometer. CD8+ T cells were further isolated from single cell suspension using mouse CD8a+ T Cell Isolation Kits (Miltenyi Biotec). Isolated CD8+ T cells were stained and analyzed as described above.
Anti mouse cd16 32 antibody clone 93
Manufactured by BioLegend
Sourced in United States
Anti-mouse CD16/32 antibody (clone 93) is a lab equipment product that binds to the mouse CD16 and CD32 receptors. It can be used for flow cytometry and other immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Cell activation cocktail with brefeldin a, by BioLegend (3 mentions)
Brefeldin a, by BioLegend (2 mentions)
Mouse tumor dissociation kit, by Miltenyi Biotec (1 mentions)
7 aad, by BioLegend (1 mentions)
Gentlemacs octo dissociator, by Miltenyi Biotec (1 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Vazyme (1 mentions)
Percp conjugated anti cd45 antibody clone 30 f11, by BioLegend (1 mentions)
Novocyte, by Agilent Technologies (1 mentions)
Facsdiva software, by BD (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Apc cy7 conjugated anti mouse cd45, by BD (1 mentions)
Fitc conjugated anti mouse cd11b, by BioLegend (1 mentions)
Fitc conjugated anti mouse cd8a, by BioLegend (1 mentions)
Apc conjugated anti mouse gr1, by BioLegend (1 mentions)
Pe conjugated anti mouse cd4, by BD (1 mentions)
Bx51wi microscope, by Olympus (1 mentions)
Act 1, by Nikon (1 mentions)
12 protocols using anti mouse cd16 32 antibody clone 93
1
Flow Cytometry Analysis of Cells
2
Multicolor Flow Cytometry of Immune Cells
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Cells were washed with the buffer, fixed, and permeabilized with FoxP3/Transcription Factor Staining Buffer Set (eBioscience/Thermo Fischer Scientific) to stain the intracellular markers. Harvested cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM) with a cell activation cocktail with brefeldin A (Biolegend) for 4 hours at 37°C. The cells were stained with the cell surface antibodies and intracellular marker in the buffer with brefeldin A. Anti-mouse CD16/32 antibody (clone 93, Biolegend, San Diego, California, USA) was added for FcR blockade and incubated for 5min at room temperature. After another washing step, antibodies for cell phenotyping were added, and cells were incubated for 40min at room temperature. The monoclonal antibodies used for flow cytometry analysis were CD8a (5H10-1), FoxP3 (FJK-16s), Cxcr3 (CXCR3-173), IFN-γ (XMG1.2), CD45 (30-F11), CD3e (145-2C11) and CD4 (RM4-5). The gating strategy of flow cytometry analysis is shown in Fig. S11 .
3
Hepatic Nonparenchymal Cells Phenotyping
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Briefly, 106 hepatic NPCs were incubated with anti-mouse CD16/32 antibody (clone 93, Biolegend, San Diego, CA, USA) for 10 min to minimize nonspecific binding. Cells were incubated with a PerCP-conjugated anti-CD45 antibody (clone 30-F11, Biolegend), an APC-labeled anti-F4/80 antibody (clone BM8, Biolegend), a FITC-conjugated anti-CD11b antibody (clone M1/70, Biolegend), a PE-Cyanine7-conjugated anti-Ly6C antibody (clone HK1.4, Biolegend), and a PE-conjugated anti-CCR2 antibody (clone SA203G11, Biolegend) or incubated with a PerCP-conjugated anti-CD45 antibody (clone 30-F11, Biolegend), an APC-conjugated anti-CD3 antibody (clone 17A2, Biolegend), a PE-Cyanine7-conjugated anti-NK1.1 (clone PK136, Biolegend), a PE-conjugated anti-CD4 (clone GK1.5, Biolegend), and a FITC-conjugated anti-CD8a (clone 53-6.7, Biolegend). The results were collected by a BD FACSAria III Flow Cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo 10.4 software (BD Bioscience).
After the cells were treated with hypoxia-reoxygenation (H/R), apoptosis was assessed by an Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme Biotech, Nanjing, China) based on the manufacturer’s protocols.
After the cells were treated with hypoxia-reoxygenation (H/R), apoptosis was assessed by an Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme Biotech, Nanjing, China) based on the manufacturer’s protocols.
4
Quantifying Cell Surface Markers
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Following spontaneous or CCL19-directed migration in a collagen matrix cells were incubated for 5 min with R10 medium containing 1:100 diluted anti-mouse CD16/32 antibody (clone 93, biolegend) in order to prevent non-specific binding of immunoglobulins to the Fc receptors prior to incubation for 20 min with AF647 anti-mouse I-A/I-E antibody (clone M5/114.15.2, biolegend) diluted 1:100 in R10 medium containing anti-mouse CD16/32 antibody. Cells were washed with R10 medium by flushing across the migration chamber. Quantification of the staining intensity was performed in Fiji. Cell outlines were drawn on the bright-field image and then applied to the fluorescent image. Background subtraction was performed locally for every cell. Corrected total cell fluorescence (CTCF) was calculated as follows: Integrated Density–Area of selected cell * mean fluorescence of background readings).
5
Labeling, Isolation, and Analysis of Lymph Nodes
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DiD-labeled LPs were injected into the foot pad of the LFM model. The ILNs and ALNs were resected at 24 h after the administration. LNs were digested in 0.1 mg/mL collagenase IV, 0.2 mg/mL collagenase D, and 0.1 mg/mL DNase I in RPMI 1640 (with 1 v/v % FBS) for 30 min. These were reliable procedures performed in the previous several reports.38 (link), 39 (link), 40 (link) The cell suspensions were washed twice with 0.5% BSA/0.1% sodium azide in PBS (fluorescence-activated cell sorting [FACS] buffer). After dispersing the cell suspensions with a 70-μm cell strainer, 2.0 × 106 cells were incubated in 10 μg/μL of anti-mouse CD16/32 antibody (clone: 93; BioLegend). Cells were stained and analyzed with a Novocyte (ACEA Biosciences, San Diego, CA, USA) according to Figure S7 .
6
Flow Cytometry Immunophenotyping Assay
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Single-cell suspensions were incubated with anti-mouse CD16/32 antibody (clone 93; Biolegend, San Diego, CA) for 5 minutes prior to staining for immune cell markers for 15 minutes at room temperature. The following monoclonal antibodies were used: APC-CY7 conjugated anti-mouse CD45 (BD Biosciences Cat:557659 Clone:30-F11), FITC conjugated anti-mouse CD11b (Biolegend Cat:101205 Clone:M1/70), APC conjugated anti-mouse Gr1(Biolegend Cat:108412,Clone: RB6-8C5), FITC conjugated anti-mouse CD8a(Biolegend Cat:100706 Clone:53-6.7), PE conjugated anti-mouseCD4(BD Biosciences Cat:553048 Clone: RM4-5), APC conjugated anti-mouse PD-L1(Biolegend Cat:124311 Clone: 10F.9G2), and IFN-γ (XMG1.2).The flow cytometry analyses were performed using a BD Fortessa Flow Cytometer (BD Fortessa). BD FACS Diva software V.5.0.1 (BD) or Flow Jo (Tree Star) was used for data processed. For cytokine staining, harvested cells were incubated in RPMI-1640 medium with cell activation cocktail with brefeldin A (Bio legend) for 6 hours at 37°C, and stimulated cells were stained as described above.
7
Lung Tissue Preparation and Imaging
8
Immunophenotyping of Vaginal and Uterine Cells
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The vaginal and uterine inflammatory cells were blocked with purified an anti-mouse CD16/32 antibody (clone 93; Biolegend), an Fc-receptor blocking antibody, and stained with various fluorochrome-conjugated secondary antibodies (Biolegend; listed in Table 1 ) or mouse CD1d tetramer-PE (Medical & Biological Laboratories). The immune cells were enumerated using the FACS Canto II® Cell Analyzer (BD Biosciences). These data were analyzed using FlowJo, version 10.6.1 (Tree Star, Inc, Ashland, OR, USA).
9
Phenotypic Analysis of Mucosal B Cells
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A week after the last immunization, the NALT and nasal passage were harvested from the upper jaw of the mice. Mononuclear cells from NALT were obtained by scraping the NALT or nasal passage tissue. After treatment with ACK lysis buffer to remove red blood cells, the cells were suspended in a staining buffer (PBS containing 2% heat-inactivated fetal bovine serum and 0.1% sodium azide) and then treated with anti-mouse CD16/32 antibody (clone 93; BioLegend) for 30 min at 4 °C to block Fc receptors. After washing with staining buffer, the cells were stained with phycoerythrin (PE)-conjugated rat anti-mouse B220 (clone RA3-6B2; BD Bioscience, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse IgA (clone C10-3; BD Bioscience), or PE-conjugated rat anti-mouse CD138 (clone 281-2; BD Bioscience) for 30 min at 4 °C. After washing with staining buffer, the cells were incubated with 7-amino-actinomycin D (7-AAD; BioLegend) for 10 min at 4 °C and then analyzed using a FACSCalibur instrument (BD Bioscience).
10
Characterization of Lymph Node Responses to Immunization
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Mice were administered 3 μg of OVA in PBS containing 30% HP-β-CyD and 10 μg of K3 CpG-ODN at the base of the tail. After 24 h, the draining lymph node was collected, and the weight and number of cells were measured. Then, the cells were incubated with anti-mouse CD16/32 antibody (clone 93; Biolegend) to block of Fc receptors and stained with the following antibodies from Biolegend: anti-mouse CD11c (clone N418), Siglec-H (clone 551), CD19 (clone 6D5), CD40 (clone 3/23), CD69 (clone FN50), CD86 (clone GL-1), and DEC205 (clone NLDC-145). Dead cells were detected using 7-AAD (eBioscience, San Diego, CA, USA) or Live/Dead™ fixable blue dead cell stain kit (Invitrogen, Life Technologies, Carlsbad, CA, USA) and excluded from the analysis. Data were obtained with a BD Accuri C6 or BD LSR II flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed by BD Accuri C6 software (BD Bioscience) or FlowJo software (Tree Star, Ashland, OR, USA).
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