Tumor and surrounding regions were delineated macroscopically. Tissues were dissociated through 100 µm cell strainers in PBS with 3% bovine serum albumin (BSA). Hepatocytes were removed by centrifugation on a 35% Percoll gradient at 700 × g at 21°C for 12 min. Leukocytes present in the pellet were resuspended in red blood cell lysis buffer (0.15 M NH4Cl, 0.1 mM KHCO3, 0.1 mM Na2-EDTA in water; pH 7.2) for 1 min, washed, and then resuspended in PBS with 3% BSA. Cell suspensions were incubated with anti-mouse CD16/CD32 (Becton Dickinson, BD, USA) to block Fc receptors and Fixable Viability Dye eFluor 506 (eBioscience, San Diego, CA, USA). Cells were then stained with a cocktail of directly conjugated mAbs [CD3 (17A2) BD, CD4 (RM4-5) eBioscience, CD8 (53-6.7) BioLegend, CD45 (30-F11) BioLegend and Foxp3 (NRRF-30) eBioscience] for 30 min at 4°C. Intracellular staining was performed with a transcription factor staining buffer set from (eBioscience). The relevant fluorescence-minus-one labeling conditions including the appropriate isotype-matched mAb were used as controls. All samples were acquired on an LSR Fortessa flow cytometer (BD) and analyzed with FlowJo version 9.3.1 or above (Tree Star, Ashland, OR, USA).
Cd45 30 f11
Manufactured by BioLegend
Sourced in United States
CD45 (30-F11) is a mouse monoclonal antibody that recognizes the pan-leukocyte antigen CD45. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of all nucleated hematopoietic cells and plays a critical role in lymphocyte activation and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b m1 70, by BioLegend (21 mentions)
Ly6g 1a8, by BioLegend (13 mentions)
Ly6c hk1, by BioLegend (12 mentions)
Cd11c n418, by BioLegend (11 mentions)
Cd8 53 6, by BioLegend (9 mentions)
Cd19 6d5, by BioLegend (9 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (8 mentions)
F4 80 bm8, by BioLegend (7 mentions)
Cd4 gk1, by BioLegend (7 mentions)
Cd3 17a2, by BioLegend (6 mentions)
Facsdiva software, by BD (5 mentions)
Cd11b m1 70, by Thermo Fisher Scientific (5 mentions)
Nk1.1 pk136, by BioLegend (5 mentions)
Cd103 2e7, by BioLegend (5 mentions)
Cd3 145 2c11, by BioLegend (5 mentions)
Lsrfortessa, by BD (5 mentions)
Facscanto 2, by BD (5 mentions)
Facsaria 2, by BD (5 mentions)
Cd62l mel 14, by BioLegend (4 mentions)
Cd69 h1.2f3, by BioLegend (4 mentions)
72 protocols using cd45 30 f11
1
Tumor Immune Cell Profiling
2
Immunophenotyping of Thymic Subsets
3
Multiparametric Flow Cytometry Profiling
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Cell suspensions from spleen, lymph nodes, and salivary glands were incubated with the fluorophore-labeled antibodies for 45 min on ice. For intracellular labeling cells were first labeled with surface antibodies and then fixed/permeabilized with the Foxp3 staining buffer set (eBioscience) and finally stained with antibodies against intracellular antigen. The anti-mouse monoclonal antibodies B220 (RA3-6B2), CD19 (6D5), CD5 (53-7.3), CD138 (281-2), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD3 (17A2), Ly9 (Ly9ab3), integrin beta 7 (FIB504), and CD45 (30-F11) were purchased from BioLegend; GL7 (GL-7), T-Bet (eBio4B10), PLZF (Mags.21F7), CD62L (MEL-14) and CD93 (AA4.1) were from eBioscience; CD23 (B3B4), CD95 (Jo2), RORγT (Q31-378), CD44 (IM7), and CD45RB (16A) were from BD Bioscience; CD49d (R1-2) was from Milteny Biotech; and goat anti-mouse IgM polyclonal antibody from Southern Biotech. Finally, PBS57-loaded mCD1d tetramer was kindly provided by the NIH Tetramer Core Facility. Data were acquired with LSRII Fortessa or FACSCanto II flow cytometers (BD Biosciences) and analyzed with FlowJo vX.0.7 (Tree Star, Inc) software. Flow cytometry experiments were performed as described (23 (link)).
4
Multiparameter Flow Cytometry of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were collected in 20 mM EDTA in Eppendorf tubes. PBMCs were spun for 8 minutes at 10,000 rpm at 20°C and serum was removed. RBCs were lysed with ACK Lysis buffer (GIBCO) for 8 minutes at room temperature (rt). If incomplete lysis occurred, cells were spun down for a second time at 12,000 rpm for 30 s and the ACK lysis step was repeated. Mononuclear cells were stained 1:100 with CB101–109/H-2Db-PE tetramer in the presence of Fc block 1:100 (αCD16/32, Tonbo), and monoclonal antibodies at 1:100 specific to CD45 (30-F11, Biolegend), CD8α (53–6.7, ebioscience), LFA-1 (clone H155–78, Biolegend), CD44 (clone IM7, BD), CD62L (MEL-14, Biolegend), PD-1 (J43, Invitrogen), KLRG1 (2F1, Biolegend), Tim-3 (RMT3–23, Biolegend), Lag-3 (C9B7W, Biolegend). CD19 (1D3, BD), F4/80 (T45–2342, BD), and CD11c (N418, BD) were used for a dump channel along with ghost dye BV510 at 1:500 (Tonbo). Antibodies were diluted in FACs buffer (PBS + 2.5% FBS) and cells were stained at room temperature in the dark for 60 minutes and then added to 100 μl of cell counting beads (Sigma Aldrich). Stained cells were fixed with 0.4% paraformaldehyde and stored overnight in the dark at 4°C prior to FACs acquisition using a Fortessa 1770 flow cytometer and Facs Diva software (BD Biosciences) and analyzed using FlowJo software (version 10).
5
Multiparametric Flow Cytometry of Liver Immune Cells
6
Multiparameter Flow Cytometry of Tumor Cells
7
Multiparametric Flow Cytometry Analysis
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Data were acquired on LSR Fortessa or LSRII flow cytometers (BD Biosciences) and analyzed using FlowJo and softwares designed by the Division of Computer Research and Technology, NCI. Live cells were gated using forward scatter exclusion of dead cells stained with propidium iodide. The following antibodies were used for staining: TCRβ (H57-597), B220 (RA3-6B2), NK1.1 (PK136), CD11b (M1/70), CD44 (IM7), CD103 (2E7), and CD69 (H1.2F3), all from eBioscience; CD3 (2C11), CD4 (GK1.5), CD8α (53-6-7), TCRγδ (GL3), and CD11c (HL3), all from BD Biosciences; and CD45 (30-F11), CD8β (YTS156.7.7), and Ly6C (HK1.4) from BioLegend. CD1d tetramers loaded with PBS-57 and unloaded controls were obtained from the NIH tetramer facility (Emory University, Atlanta, GA, USA).
8
Characterization of Th1/Th2/Th17 Cytokines and Immune Cell Populations
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To label the Th1 and Th2 cytokines in serum, we used a Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine kit (BD Biosciences) following the manufacturer's instructions and using a FACS Canto II (BD Biosciences). To characterize the immune cell populations, single cell suspensions of lymphocytes were prepared from large intestine lamina propria
34 , 35 . Lymphocytes were stimulated for 4 h using ionomycin, phorbol myristate acetate (Sigma), and 1 μg/ml Golgistop (Sigma). Prior to flow cytometry, lymphocytes were stained with fluorescent conjugated antibodies against CD4 (RM‐4‐5, 1/800), Ifng (XMG1.2, 1/200), IL‐13 (eBIO 13a, 1/200), TCRa/b (MR5.2, 1/200) (all Fisher Scientific), CD45 (30‐F11, 1/200), CD8 (53‐6.7, 1/200), IL‐4 (11B11, 1/200) (all Biolegend, London, UK), IL‐17 (TC11‐18H10.1, 1/100) and TNF (MP6‐XT22, 1/200) (both BD Biosciences), and a live/dead marker. Data were analysed using FCAP Array v3.0 (BD Biosciences) and FlowJo (FlowJo LLC, Ashland, OR, USA) software. Statistical analysis was performed using three to five animals per genotype and three independent experiments. Significance was calculated using a three‐way full factorial fit model and a joint F‐test to assess the effects of genotype, treatment, and experiment day on the cytokine response
36 .
9
Isolation and Characterization of Colonic Immune Cells
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Lamina propria cells from colonic tissue were harvested as before8 (link). For intracellular cytokine staining, cells were stimulated using the eBioscience cell stimulation cocktail for 4 h at 37 °C. Cells were fixed and permeabilized using the Biolegend fixation and permeabilization buffer. The following antibodies (clones) were used for staining: CD45 (30-F11), CD19 (6D5), CD11b (M1/70), CD90.2 (53–2.1), CD3 (145–2C11), TCR-β (H57–597), CD4 (RM4–5), CD8 (53–6.7), Gr-1 (RB6–8C5), MHCII (M5/114.15.2), CD11c (N418), CD103 (2E7), IFN-γ (XMG1.2) from Biolegend and IL-22 (1H8PWSR), and IL-13 (eBio13A) from eBioscience. All samples were blocked with Fc block (TruStainfcx) and stained with a fixable live dead stain (Invitrogen). FlowJo v.10 was used to analyze flow cytometry data. Intestinal epithelial cells (IECs) were harvested by incubation at 37°C with 2mM DTT (Sigma) followed by two incubations with 5mM EDTA and then digested with Dispase (Sigma) and DNase (Sigma). IECs were sorted as PI−CD45−EpCam+ using the following antibody clones: CD45 (30-F11) and EpCam (G8.8) from Biolegend on an FACSAria II (BD Biosciences). Purity of IECs was ≥ 95%.
10
Comprehensive Immune Cell Profiling
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Single cell suspensions were stained with fixable viability dye eFluor® 780 (eBioscience) for 30 min at 4°C, and Fc receptors were blocked with anti-CD16/32 (BioLegend) blocking antibody prior to surface staining with antibodies. Antibodies for surface staining used are listed, mouse: CD45 (30-F11, BioLegend), TCR β (H57-597, BioLegend), CD69 (H1.2F3, eBioscience), CD1d tetramer (NIH); human: CD45 (HI30, eBioscience), TCRα/β (IP26, BioLegend), CD69 (FN50, BioLegend), Vα24 (6B11, BD Bioscience). For intracellular staining, single cell suspensions were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 1 μg/mL Golgi stop A (BD Biosciences) for 4 h. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD biosciences) and further stained intracellularly with human IFNγ (B27, BioLegend) and human IL-4 (MP4-25D2, BioLegend). For Ki67 staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (eBioscience), and further stained with Ki67 (SolA15, eBioscience). Data were acquired using LSR II (BD Biosciences) and analyzed using the FlowJo software (BD Biosciences).
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