Stromal cells were collected by trypsin and prepared by passing cells through a 40-µm cell strainer. The resulting single-cell suspensions were stained in FACS buffer with the following antibodies for flow cytometric analysis: anti-mouse CD3 (BD Bioscience), and anti-mouse CD45, CD31, Podoplanin, LTβR, VCAM-1 (All from BioLegend). TLSs were dissected from mice, mechanically dissociated and digested with tumor digestion buffer and GentleMACS (Miltenyi Biotec). After lysis of RBCs, the single-cell suspensions were analyzed by flow cytometry with the following antibodies: anti-mouse CD3, CD4, CD11b, CD11c (All from BD Bioscience), anti-mouse CD8, CD19, CD45, CD31, Podoplanin (All from BioLegend), and anti-NK1.1 (eBioscience). MC38 tumors were processed as above and stained with the following antibodies: anti-mouse CD3, CD4, CD69, CD27, CD45RA, PD-1, LAG3, and CD127 (All from BD Bioscience), and anti-mouse CD8, CD62L, CD44, KLRG1, CTLA-4, Tim-3, and CD45.2 (All from BioLegend). DAPI (Sigma-Aldrich) was used as a cell viability marker. The cells were analyzed by the LSR II flow cytometry equipped with five lasers (BD Biosciences), and the data were analyzed with Flow Jo (Tree Star).
Anti mouse cd3
Manufactured by BD
Sourced in United States
Anti-mouse CD3 is a laboratory reagent used in flow cytometry applications. It targets the CD3 antigen, which is expressed on the surface of mouse T cells. This product can be used to identify and quantify T cell populations in mouse samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse cd28, by BD (8 mentions)
Anti mouse cd4 microbeads, by Miltenyi Biotec (6 mentions)
Anti mouse cd28, by Thermo Fisher Scientific (5 mentions)
Anti mouse cd4, by BD (5 mentions)
Anti ifn γ, by BD (5 mentions)
Anti mouse cd19, by BD (4 mentions)
Retinal, by Merck Group (4 mentions)
Soluble anti mous cd28, by BD (4 mentions)
Human recombinant transforming growth factor β tgf β, by Thermo Fisher Scientific (4 mentions)
Anti human cd4 microbeads, by Miltenyi Biotec (4 mentions)
Anti il 4, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
F ab 2 fragment goat anti mouse igm, by Jackson ImmunoResearch (3 mentions)
Concanavalin a (cona), by Merck Group (3 mentions)
Topcount nxt, by PerkinElmer (3 mentions)
3h thymidine, by PerkinElmer (3 mentions)
Anti mouse cd8, by BD (3 mentions)
Cd11b, by BD (2 mentions)
Ifn γ, by BD (2 mentions)
Anti mouse cd45, by BioLegend (2 mentions)
50 protocols using anti mouse cd3
1
Multiparametric Flow Cytometry Analysis of Tumor Immune Microenvironment
2
Splenocyte IFNγ Assay for Antigen-Specific T-Cell Response
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Spleens used in step 2.5 were used in ICS assays: one million splenocytes/well were incubated with 2 μg/mL of HBsAg-specific peptide pools (15mer overlapping by 11, custom order JPT peptide solutions, Germany) or equal quantity of DMSO in the presence of brefeldin A and anti-mouse CD28 and CD48b antibodies (BD Biosciences) for 6 h at 37 °C. Splenocytes were surface stained for anti-mouse-CD3 (BD cat #557596), CD4 (BD cat #550954), CD8 (BD cat #563068), permeabilized and stained for anti-mouse IFNγ (BD cat #557596). OVA affected splenocytes were incubated with 1 µg/mL of OVA major histocompatibility complex (MHC) class-I epitope peptide 257-264 (SIINFEKL) or OVA MHC class-II epitope peptide 323-339 (ISQAVHAAHAEINEAGR) purchased from Invivogen, San Diego, CA for 5 h at 37 °C. Splenocytes were surface stained for anti-mouse-CD3(#557596), CD4(#550954), CD8(#563068), permeabilized, and stained for anti-mouse IFNγ (BD cat #557596). A FACS LSRII flow cytometer read fixed samples (BD Biosciences), and data was analyzed using FloJo software (Treestar Inc., Ashland, OR, USA).
3
Immunogenic Epitopes and Cytokine Profiling
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Gal-GalNAc (ZOM-TRI-017) was purchased from Omicron, USA. The sequences of Fap2 and FadA immunogenic epitopes were “TELAYKHYFGT” and “MKKFLLLAVLAVSASAFA”, respectively, and were composed by Abiocenter, China. The carcinogen azoxymethane (AOM, A5486) was purchased from Sigma, USA. Anaerobe basal broth (CM0957B) and anaerobe basal broth plates (CM0972B) were purchased from Oxoid, UK. RPMI 1640(11875101), DMEM (No Glu, 11966025), trypsin 0.25% solution (15050065), fetal bovine serum (FBS; 10099), and PBS buffer (10010031) were purchased from Gibco, USA. Murine MCSF (315-02) and interleukin (IL)-4 (214-14) were purchased from PEPROTECH. Goat anti-mouse IgG1 heavy chain (HRP) (ab97240), goat anti-mouse IgG2a heavy chain (HRP) (ab97245), and goat anti-mouse IgG2b heavy chain (HRP) (ab98703) antibodies were purchased from Abcam. A mouse IL-1β (432604), mouse IL-4 (431107), mouse IL-6 (431307), mouse IL-12p40 (431604), mouse IL-17A (432507), mouse IFN-γ (430807), and mouse TNF-α ELISA kits (430907) were purchased from Biolegend, USA. A mouse IFN-γ EliSpot kit (CT317-PR20) was purchased from Dakewe, China, and a mouse IL-17 ELISpot kit (A42927) was purchased from eBioscience. The anti-mouse-CD3, CD4, CD8, IFN-γ, CD11b, Ly-6C and Ly-6G fluorescent antibodies were purchased from BD Biosciences, CA, USA.
4
Arg-Dependent T and B Cell Activation
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Splenic lymphocytes (2 × 105 cells/well) from naïve Arg+/+ and Arg−/− mice were stimulated in vitro with purified anti-mouse CD3 (BD Biosciences, San Jose, CA) and anti-mouse CD28 (eBioscience, San Diego, CA), Concanavalin A (ConA) (Sigma-Aldrich, St. Louis, MO), lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO), and F(ab’)2 Fragment Goat Anti-Mouse IgM (Jackson Immuno Research, West Grove, PA). Cells were stimulated for 72 hours, and pulsed with [3H]- thymidine (1 μCi/well) (Perkin Elmer, Waltham, MA) for the last 16–18 hours. Cells were harvested and [3H]-thymine incorporation monitored on a TopCount NXT (Perkin Elmer, Waltham, MA).
5
Alloantigen-Induced Lymph Node Expansion
6
Isolation of B Cell Subsets from Peritoneal Cells
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Peritoneal cells were stained with antibodies in sorting buffer (1 mM EDTA, 25 mM HEPES pH 7.0, 1% FBS diluted in PBS). To isolate the B cells, the peritoneal cells were incubated with anti-mouse CD19 (BD Biosciences, San Jose, CA, USA) and anti-mouse CD23 (eBioscience, San Diego, CA, USA). To sort non-B cells from the peritoneal cells, anti-mouse CD3 (BD Bioscience) was used to isolate T cells. B1 cells and B2 cells were sorted using a FACSAria™ II system (Becton Dickinson Inc.).
7
Characterizing MSC Surface Markers
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The expression of surface markers on MSCs was
investigated using the following antibodies. Anti-mouse
CD34 (PE, eBiosience, USA), anti-mouse CD44 (APC,
BD, USA), anti-mouse CD45 (APC-cy7, Biolegend,
USA), anti-mouse CD73 (PE, BD, USA), anti-mouse
CD90 (APC, BD, USA), anti-mouse CD105 (PE,
eBiosience, USA), anti-mouse Sca1 (FITC, Biolegend,
USA), anti-mouse CD3 (PE, BD, USA), anti-human
CD90 (APC, Biolegend, USA), anti-human CD105 (APC,
Biolegend, USA), anti-human CD29 (PE, eBiosience,
USA), anti-human CD45 (FITC, Biolegend, USA) and
anti-human CD34 (PE, eBiosience, USA). Passage 2 cells
were used for the analysis of cell surface markers by flow
cytometry (FACS calibur, Becton Dickinson, USA). For
flow cytometry analysis, 10,000 events were counted and
data were analyzed using the flowJo software.
investigated using the following antibodies. Anti-mouse
CD34 (PE, eBiosience, USA), anti-mouse CD44 (APC,
BD, USA), anti-mouse CD45 (APC-cy7, Biolegend,
USA), anti-mouse CD73 (PE, BD, USA), anti-mouse
CD90 (APC, BD, USA), anti-mouse CD105 (PE,
eBiosience, USA), anti-mouse Sca1 (FITC, Biolegend,
USA), anti-mouse CD3 (PE, BD, USA), anti-human
CD90 (APC, Biolegend, USA), anti-human CD105 (APC,
Biolegend, USA), anti-human CD29 (PE, eBiosience,
USA), anti-human CD45 (FITC, Biolegend, USA) and
anti-human CD34 (PE, eBiosience, USA). Passage 2 cells
were used for the analysis of cell surface markers by flow
cytometry (FACS calibur, Becton Dickinson, USA). For
flow cytometry analysis, 10,000 events were counted and
data were analyzed using the flowJo software.
8
Culturing and Transfecting Jurkat T Cells
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E6.1 Jurkat T cells were maintained at 37°C in Iscove’s modified Dulbecco’s media (Thermo Fisher Scientific) supplemented with 10% FBS (Sigma-Aldrich), sodium pyruvate, l -glutamine, penicillin-streptomycin, and MEM nonessential amino acids solution (Thermo Fisher Scientific). Transfections were performed with 1.0 × 106 cells/ml and 2 to 3 µg plasmid DNA by nucleofection using Amaxa kit V (Lonza). Primary mouse T cells were obtained via dissection of mouse inguinal, axillary, brachial, and mesenteric lymph nodes. CD8+ T cells were isolated via negative selection using a CD8a+ T cell isolation kit (Kit II, MACS; Miltenyi Biotec) and grown in DMEM supplemented with 5% FBS, l -glutamine, and penicillin-streptomycin. The cells were then activated for 72 h with plate-bound 2.5 µg/ml anti-mouse CD3 and 1 µg/ml anti-mouse CD28 antibodies (BD), followed by stimulation with 50 U/ml recombinant interleukin-2 (R&D Systems) every 48 h.
9
Quantifying LAIR-1 Blocking Capacity
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To assess the blocking capacity of NC410, a titration assay with LAIR-1 reporter cells was performed as previously described (Lebbink et al., 2006 (link)). Black Falcon clear flat bottom 96-well plates were coated with 5 μg/mL human collagen I (Sigma-Aldrich) in 2 mM acetic acid (Merck), anti-mouse-CD3 (BD), anti-human-LAIR-1 antibody (clone 8A8) in PBS (Sigma-Aldrich) or isotype control (eBioscience) in PBS by spinning down for 3 min at 1700 rpm and incubating overnight at 4°C. The next day, plates were washed with PBS and pre-incubated with the indicated concentrations of NC410 or isotype control (NextCure) in culture medium by spinning down for 5 min at 1500 rpm at room temperature (RT) and incubating for 2 hr at 37°C.
WT and hLAIR-1 reporter cells were harvested and seeded at 1 × 106 cells/mL in 50 μL/well on top of the collagen and fusion protein-treated wells and spun down for 3 min at 1700 rpm at RT. Plates were incubated overnight, approximately 16 hr, at 37°C, and GFP expression was measured on a LSRFortessa (BD Biosciences).
WT and hLAIR-1 reporter cells were harvested and seeded at 1 × 106 cells/mL in 50 μL/well on top of the collagen and fusion protein-treated wells and spun down for 3 min at 1700 rpm at RT. Plates were incubated overnight, approximately 16 hr, at 37°C, and GFP expression was measured on a LSRFortessa (BD Biosciences).
10
Multiparametric Immune Phenotyping
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Surface cell staining was performed with anti-mouse CD3, CD4, CD25, CD8, CD62L, and CD44 mAbs (all from BD Pharmingen) following a 2.4G2 Fc block (anti-CD16/CD32 mAb) step. FOXP3 expression was tested with the FOXP3 staining kit (eBioscience, San Diego, CA) according to manufacturer’s instructions. Cells were acquired on a FACSCanto (BD Bioscience) and analyzed with FlowJo (Tree Star, USA) software.
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