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39 protocols using anti mouse cd45

1

Quantifying Antigen-Specific Thymocytes

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Mouse thymi were cut into 8 μm frozen sections using a Leica CM1520 cryostat (Leica Biosystems). Anti-mouse Gr1 and anti-mouse CD45.2 were purchased from BioLegend. Anti-mouse cytokeratin 5, anti-mouse cytokeratin 8, anti-HBsAg, and anti-HBsAg were from Bioss Inc. H-2Ld HBsAg28-39 (IPQSLDSWWTSL) MHC-I tetramer (PE) was used to detect HBsAg-specific CD8+ thymocytes in the immunofluorescence (IF) test.
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2

Isolation and Characterization of Murine Myeloid Cells

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Bone marrow was harvested from femurs and red blood cells were lysed (RBC lysis buffer, Biolegend). Bone marrow cells were blocked with TruStain FcX (Clone 93, Biolegend) and stained with anti-mouse CD45.2 (Clone 104, Biolegend) or IgG2a κ Isotype (Clone MOPC-173, Biolegend), anti-mouse CD11b (Clone M1/70, Biolegend) or IgG2b, κ Isotype (Clone RTK4530, Biolegend), anti-mouse Ly6G (Clone 1A8, Biolegend) or IgG2a, κ Isotype (Clone RTK2758, Biolegend) and anti-mouse Ly6C (Clone HK1.4, Biolegend) or IgG2c, κ Isotype (Clone RTK4174, Biolegend). Samples were analysed using a BD FACS Canto II and FlowJo software.
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3

Adoptive Transfer of Human Myeloid Cells

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BALB/c.Rag2tmlFwa IL2rgtmlWjl (Rag2−/−γc−/−) mice were bred and maintained in the animal facility of Centro de investigación médica aplicada (CIMA), Universidad de Navarra maintained at CIMA under the guidelines of the Ethics Committee of the center. CHO/mEGFR_mPSMA cells were detached and prestained with CellTracker Green CMFDA Dye (ThermoFisher Scientific; #C7025, 1:1500 dilution) according to manufacturer’s protocol for cells in suspension. The mice were intraperitoneally injected with 1.5 × 106 CMFDA-prestained CHO/mEGFR_mPSMA cells into the left side of the peritoneal cavity. Straight after, they received a separate injection into the right side of the peritoneal cavity containing 3 × 106 human CD11b+ cells isolated from a fresh buffy coat as described previously, together with 50 μg of indicated bispecific antibodies. After 24 h, we performed the peritoneal lavage with 3 ml of PBS, and the cell populations were quantified by flow cytometry on CytoFLEX S (BD; B75408). The staining included antimouse CD45.2 (Biolegend #109820, 1:200) in order to exclude murine leukocytes, antihuman CD11b PerCP/Cy5.5 (Biolegend #301328, 1:100) for effector population detection, and Zombie NIR (Biolegend #423105, 1:1000) for live/dead. The gating strategy is shown in Fig. S2.
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Isolation and Characterization of Murine Myeloid Cells

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Bone marrow was harvested from femurs and red blood cells were lysed (RBC lysis buffer, Biolegend). Bone marrow cells were blocked with TruStain FcX (Clone 93, Biolegend) and stained with anti-mouse CD45.2 (Clone 104, Biolegend) or IgG2a κ Isotype (Clone MOPC-173, Biolegend), anti-mouse CD11b (Clone M1/70, Biolegend) or IgG2b, κ Isotype (Clone RTK4530, Biolegend), anti-mouse Ly6G (Clone 1A8, Biolegend) or IgG2a, κ Isotype (Clone RTK2758, Biolegend) and anti-mouse Ly6C (Clone HK1.4, Biolegend) or IgG2c, κ Isotype (Clone RTK4174, Biolegend). Samples were analysed using a BD FACS Canto II and FlowJo software.
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5

Comprehensive Immune Cell Profiling

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The following antibodies were used for the present study.
Anti-mouse CD3, clone number 17A2, BioLegend, 05112-40-100, V450;
Anti-mouse CD4, clone number GK1.5, BioLegend, 100434, PerCP/Cyanine5.5;
Anti-mouse CD8, clone number 53-6.7, BioLegend, 100714, APC/Cyanine7;
Anti-mouse CD25, clone number 3C7, BioLegend, 101908, FITC;
Anti-mouse CD69, clone number H1.2F3, BioLegend, 104508, PE;
Anti-mouse CTLA-4, clone number UC10-489, BioLegend, 106305, PE;
Anti-mouse PD-1, clone number 29F.1A12, BioLegend, 135210, APC;
Anti-mouse CD19, clone number 1D3, eBioscience, 12-0193-82, PE;
Anti-mouse CD45, clone number 30F11, BioLegend, 103122, Alexa Fluor 488;
Anti-human CD45, clone number HI30, BioLegend, 304025, PerCP;
Anti-mouse IFNγ, clone number XMG1.2, eBioscience, PE;
Anti-mouse IL-17A, clone number TC11-18H10.1, BioLegend, Alexa Fluor 647;
Anti-mouse IL-13, clone number 13A, BioLegend, PE/Cyanine7;
Anti-mouse Foxp3, clone number MF14, BioLegend, PE;
Anti-mouse TNFα, clone number MP6-XT22, BioLegend, Alexa Fluor 488;
Purified Anti-mouse IFNγ antibody, clone number XMG1.2;
Purified anti-mouse IL-4 antibody, clone number 11B11.
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6

Alloantigen-Induced Lymph Node Expansion

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Male Balb/c mice (8-10 weeks-old) were given foot pad injections in the right hind limb of 1×107 splenocytes from male DBA-2 mice (8-10 weeks-old). Recipient mice were immediately treated as follows (schematic in Figure 8A)
On day 4 after alloantigen challenge, the mice were sacrificed and the draining ipsilateral popliteal LNs (IL-pLNs) were collected. Following mechanical disruption of the pLNs, isolated cells were counted on a Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Brea, CA, USA) to determine LN cellular expansion due to alloresponses. From each IL- pLN cell suspension, 1×106 cells were stained for flow cytometry using LIVE/DEAD fixable near infra-RED and antibodies against the following surface markers: anti-mouse CD3, anti-mouse CD8, anti-mouse CD62L (BD Bioscience), anti-mouse CD4 (e-Bioscience), anti-mouse CD44 and anti-mouse CD45 (Biolegend). Samples were evaluated using a Cytoflex flow cytometer and data analyzed with Flow Jo.
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7

Multicolor Flow Cytometry for Immune Cell Phenotyping

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Cells were stained with the relevant antibodies in FACS buffer for 30 min at 4°C. Anti-mouse antibodies used for flow cytometry staining were anti-mouse CD45 (Clone I3/2.3, Biolegend), CD19 (Clone 1D3/CD19, Biolegend), CD3 (Clone 17A2, Biolegend), F4/80 (Clone BM8, Biolegend), and Ly-6G (Clone 1A8, Biolegend). Flow cytometry was performed on BD FACSAria II and analyzed with FlowJo software (version 10.7.1, FlowJo LLC, USA).
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8

Isolation and Characterization of Murine Microglia

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Mice were trans-cardial perfused with 50 ml cold PBS containing 5 IU/ml heparin. Ischemic brains were isolated in 1 × HBSS with 25% glucose and HEPES. After thorough grinding, the total mixture passed through a 70-μm pore filter. Harvested single-cell suspensions were stratified on a 30–70% Percoll gradient (GE Healthcare BioSciences). After centrifuging at 2500 rpm at slow acceleration and deceleration for 20 min, cells at the interface were collected and stained with anti-mouse CD45 (BioLegend, 103114, 1:500), CD11b (Invitrogen, 11-0112-82, 1:500), CD68 (BioLegend, 137007, 1:300), Mertk (Invitrogen, 12-5751-82, 1:500). Among viable cells (FVS-780low), microglia (CD45intCD11b+) were isolated and analyzed by Fluorescence activated cell sorter (FACS) (BD Biosciences, Carlsbad, CA, USA).
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9

Leukocyte Isolation and Quantification

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Non-tumor bearing male mice were treated with 2.5 mg/kg of either blebbistatin or vehicle. At 3 hr after treatment, blood was collected by terminal cardiac draw, and 500 μL of blood was mixed with 10 mL of red blood cell (RBC) lysis buffer (150mM NH4Cl, 10mM NaHCO3, 1.3 mM EDTA), centrifuged at 500 g for 3 min, and resuspended in FACS buffer. Samples were blocked in FACS for 30 min and incubated with anti-mouse CD45 (1:100, BioLegend 103110) for 1 hr. Samples were washed 3 times with FACS buffer and suspended to the volume initially collected, in FACS buffer containing a specific concentration 15-μm scarlet microspheres.
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10

Flow Cytometric Analysis of Liver Immune Cells

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Mice were sacrificed and the liver was harvested for analysis ex vivo as previously described (Park et al., 2017 (link)). Cells were isolated and stained with anti-mouse CD45 (1:100 dilution, Biolegend, #103116), anti-mouse F4/80 (1:100 dilution, Biolegend, #123108), anti-mouse CD206 (1:100 dilution, Biolegend, #141720), and anti-mouse CD11c (1:100 dilution, Biolegend, #117310), and then tested using flow cytometry (LSRFortessaTM cell analyzer, BD, United States). Data were analyzed with FlowJo V10.6.
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