Colon tissue paraffin sections (5 μm) were dewaxed and rehydrated. Antigen retrieval was performed in an ethylenediaminetetraacetic acid solution. Tissue sections were blocked in bovine serum albumin for 30 min and incubated with the following primary antibodies: anti-F4/80 (1 : 1,200, Servicebio, Wuhan, China) and anti-CD16/32 (1 : 200, TONBO biosciences, San Diego, CA, USA). Phosphate buffer saline was used to wash (x3) the tissue sections. Secondary Alexa Fluor 488 or CY3 conjugated antibodies (Servicebio, Wuhan, China) were then incubated with the tissue sections for 1 h [15 (link)]. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole for 10 min. Confocal laser scanning microscopy was used for image capture.
Anti cd16 32
Manufactured by Cytek Biosciences
Sourced in China
The Anti-CD16/32 is a flow cytometry antibody reagent that binds to the CD16 and CD32 cell surface proteins. It is commonly used for the identification and characterization of immune cells, such as natural killer (NK) cells and myeloid cells, in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (3 mentions)
Fortessa 1770 flow cytometer, by BD (2 mentions)
Cb101 109 peptide, by GenScript (2 mentions)
Mp6 xt22, by BioLegend (2 mentions)
Facsdiva software, by BD (2 mentions)
Anti f4 80, by Wuhan Servicebio Technology (1 mentions)
Anti tim 3, by BioLegend (1 mentions)
Anti tnf α, by BioLegend (1 mentions)
Anti cd3, by Cytek Biosciences (1 mentions)
Anti cd8, by BD (1 mentions)
Anti cd45, by Thermo Fisher Scientific (1 mentions)
Anti cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining kit, by Thermo Fisher Scientific (1 mentions)
Anti pd 1, by BD (1 mentions)
Anti lag 3, by Thermo Fisher Scientific (1 mentions)
Anti ki67, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ, by BioLegend (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Anti cd44, by BioLegend (1 mentions)
Anti cd62l, by BioLegend (1 mentions)
7 protocols using anti cd16 32
1
Immunofluorescence Staining of Colon Tissue
2
Profiling T Cell Cytokine Response
3
Comprehensive Immune Cell Profiling of Tumor Samples
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Tumor tissues were first minced and passed through 70 μm filters to obtain single-cell suspension. Cells were then stained with Viability Ghost Dye 510 (Tonbo Biosciences, 13-0870) for 10 min at 4 °C and blocked by anti-CD16/32 (Tonbo Bioscience, 70-0161) (1 to 100 dilution) at 4 °C for 10 min. Cells were further stained with anti-CD45 (Invitrogen, 11-0451-82), anti-CD3 (Tonbo Biosciences, 65-0031-U100), anti-CD8 (BD Pharmingen, 557654), anti-CD44 (BioLegend, 103041), anti-CD62L (BioLegend, 104424), anti-PD1 (BD Biosciences, 563059), anti-TIM3 (BioLegend, 119704), and anti-LAG3 (Invitrogen, 25223182) in staining buffer (2% FBS in PBS). For nuclear transcription factor staining, cells were permeabilized using a FoxP3/transcription factor staining kit (eBioscience, 00-5523-00) and then stained with anti-Ki67 (Invitrogen, 48-5698-82). For cytokine staining, cells were stimulated by anti-CD3/CD28 (Thermo Fisher, 11452D) at 37 °C overnight and then treated with BD GolgiPlug (BD Biosciences, 550583) at 37 °C for 5 h. After surface staining, cells were permeabilized using a BD Cytofix/Cytoperm kit (BD Biosciences, 554714) and stained with anti-IFNγ (BioLegend, 505826) and anti-TNFα (BioLegend, 506314). All antibodies were used at 1:150 dilution. Cells were then fixed with 1% paraformaldehyde and analyzed by BD FACSCelesta. Data were analyzed using BD FACSDiva and FlowJo software.
4
Mouse Lymphocyte Isolation and Staining
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The following anti-mouse antibodies were purchased from BioLegend (San Diego, CA) unless otherwise specified: purified anti-CD16/32 (Tonbo, San Diego, CA), B220, CD23, IgM (eBiosciences, San Diego, CA), CD21/35, CD4, CD8, IL-10, and Foxp3.
Lymphocytes from spleen and lymph nodes of C57BL/6J mice were harvested2 (link). Cells were incubated with purified anti-CD16/32 and surface stained then fixed. Intracellular staining was carried out using the BD Cyofix/CytopermTM kit following manufacturer’s protocol. For IL-10 analysis, lymphocytes were harvested, stimulated for 5 hours with 50 ng/ml PMA and 500 ng/ml Ionomycin with GolgiStop (contains monensin) (BD, San Jose, CA) and stained5 (link). Analysis was conducting using a BD LSRII or BD CantoII flow cytometer and FlowJo software (Tree Star Ashland, OR).
Lymphocytes from spleen and lymph nodes of C57BL/6J mice were harvested2 (link). Cells were incubated with purified anti-CD16/32 and surface stained then fixed. Intracellular staining was carried out using the BD Cyofix/CytopermTM kit following manufacturer’s protocol. For IL-10 analysis, lymphocytes were harvested, stimulated for 5 hours with 50 ng/ml PMA and 500 ng/ml Ionomycin with GolgiStop (contains monensin) (BD, San Jose, CA) and stained5 (link). Analysis was conducting using a BD LSRII or BD CantoII flow cytometer and FlowJo software (Tree Star Ashland, OR).
5
Corneal Immunohistochemistry for Neutrophils
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Whole corneas were excised and immediately embedded in optimal cutting temperature (Thermo Fisher Scientific). Frozen sections (10 µm) were mounted on Superfrost™ Plus™ slides and stored at −80°C. Before staining, the slices were placed on a 37°C hot plate for 10 min followed by submersion in acetone for 10 min at −20°C. The slides were washed in PBS twice, permeabilized 5 min in 0.1% Triton-X/PBS, and incubated at room temperature for 1 h in blocking buffer containing 2% BSA and 0.1% Tween 20 with 1:200 Fc block (Anti-CD16/32; Tonbo Biosciences), and 10% donkey serum (Vector Labs).
Corneal sections (5 µm) were then stained at room temperature for 2 h with Ly6G antibody NIMPR14 at 40 µg/ml (Abcam) and 1:50 Rabbit polyclonal antibody to histone H3 (citrulline R2 + R8 + R17) (Abcam). Slides were rinsed three times in PBS, and then secondary antibodies, donkey anti-rabbit Alexa Fluor 555 or goat anti-rat Alexa Fluor 488, were applied at 1:1,000 (Invitrogen) for 1 h at room temperature. Slides were rinsed and mounted in Fluoromount with DAPI (Thermo Fisher Scientific). All images were acquired within 24 h.
Corneal sections (5 µm) were then stained at room temperature for 2 h with Ly6G antibody NIMPR14 at 40 µg/ml (Abcam) and 1:50 Rabbit polyclonal antibody to histone H3 (citrulline R2 + R8 + R17) (Abcam). Slides were rinsed three times in PBS, and then secondary antibodies, donkey anti-rabbit Alexa Fluor 555 or goat anti-rat Alexa Fluor 488, were applied at 1:1,000 (Invitrogen) for 1 h at room temperature. Slides were rinsed and mounted in Fluoromount with DAPI (Thermo Fisher Scientific). All images were acquired within 24 h.
6
Profiling T Cell Cytokine Response
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To determine peptide-specific cytokine production, single cell suspension of spleen and tumor were cultured in complete T cell media in the presence of Golgiplug (1:500 BD Biosciences) and CB101–109 peptide (1 μg/ml Genscript) for 5 hours at 37°C. Next, cells were stained for Ghost live-dead (Tonbo), CD45, CD8α, CD44, PD-1 as described above, then fixed and permeabilized (BD Fixation Kit) and stained for intracellular expression of IFNγ (XMG1.2, Biolegend, 1:100) and TNFα (MP6-Xt22, Biolegend, 1:100) overnight in the dark at 4°C. To profile the phenotype of splenic and intratumoral T cells, mononuclear cells were stained with CB101–109/H-2Db-PE tetramer (1:100) in the presence of 1:500 Fc block (anti-CD16/32, Tonbo), and monoclonal antibodies to the following antibodies were used for surface stain (same clones as stated above): CD45, CD8, CD44, PD-1, Tim-3, Lag-3, and TIGIT (1G9, Biolegend). Following fixation with the intracellular fixation and permeabilization Foxp3 kit (Tonbo), Ki67 (B56, Biolegend) was used to stain the cells for 30 minutes in the dark on ice. Cells were washed and analyzed using a Fortessa 1770 flow cytometer and Facs Diva software (BD Biosciences) and analyzed using FlowJo software (version 10).
7
Flow Cytometry Immune Cell Profiling
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