The mice were humanely sacrificed, and the spleens were removed. The spleens were triturated, and the red blood cells were removed using RBC Lysis Buffer (Solarbio) according to the kit instructions. After centrifuging at 500 g for 10 min at 4°C, the supernatants were discarded, and cells were resuspended and washed twice with Staining buffer (eBioscience) to obtain single-cell suspensions. Then, cells were stained with different combinations of flow cytometry antibodies, including APC-conjugated anti-mouse CD3 (eBioscience, San Diego, United States), FITC-conjugated anti-mouse CD4 (BioLegend, San Diego, United States), PE-conjugated anti-mouse CD8 (eBioscience) APC-conjugated anti-mouse B220 (BioLegend), Pacific Blue-conjugated anti-mouse GL-7 (BioLegend), PE-conjugated anti-mouse CD95 (BioLegend), PE-conjugated anti-mouse PD-1 (BioLegend), and Brilliant Violet 421-conjugated anti-mouse CXCR5 (BioLegend). After staining, cells were washed with Staining buffer and dispersed in 500 mL of Staining buffer. Analysis was performed using a Mona CytoFLEX flow cytometer (BeckmanCoulter LifeSciences, Brea, United States).
Fitc conjugated anti mouse cd4
Manufactured by BioLegend
Sourced in United States
FITC-conjugated anti-mouse CD4 is a monoclonal antibody that binds to the mouse CD4 cell surface glycoprotein. CD4 is a co-receptor expressed on the surface of T helper cells and is involved in the recognition of antigen-MHC class II complexes.
Automatically generated - may contain errors
Lab products found in correlation
Staining buffer, by Thermo Fisher Scientific (3 mentions)
Apc conjugated anti mouse b220, by BioLegend (3 mentions)
Pacific blue conjugated anti mouse gl 7, by BioLegend (3 mentions)
Pe conjugated anti mouse cd95, by BioLegend (3 mentions)
Pe conjugated anti mouse pd 1, by BioLegend (3 mentions)
Brilliant violet 421 conjugated anti mouse cxcr5, by BioLegend (3 mentions)
Apc conjugated anti mouse cd3, by Thermo Fisher Scientific (3 mentions)
Apc conjugated anti mouse ifn γ, by BioLegend (3 mentions)
Pe conjugated anti mouse cd8a, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Pe conjugated anti mouse cd25, by BioLegend (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Purified anti mouse cd28 mab, by BioLegend (2 mentions)
Anti mouse cd3 mab, by BioLegend (2 mentions)
Rbc lysis buffer, by Solarbio (1 mentions)
Pe conjugated anti mouse cd8, by Thermo Fisher Scientific (1 mentions)
Cyan adp cytometer, by Beckman Coulter (1 mentions)
Fix perm kit, by BioLegend (1 mentions)
Lightning link apc conjugation kit, by Abcam (1 mentions)
Pe cy7 conjugated anti mouse f4 80, by BioLegend (1 mentions)
18 protocols using fitc conjugated anti mouse cd4
1
Spleen Cell Immunophenotyping by Flow Cytometry
2
Multiparametric Flow Cytometry Analysis
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Flow cytometry was applied to analyze the cell surface molecule expression and intracellular cytokine expression. The antibodies used were as follows: FITC-conjugatedanti-mouse CD4; F4/80; TNF-α; IFN-γ; anti-human CD14; eFluor® 488-conjugated anti-mouse TNF-α; PE-conjugated anti-mouse CD8; Tim-3; TGF-β1; IL-10; anti-human Tim-3; PerCP/Cy5.5-conjugated anti-mouse IL-17A; PE/CY7-conjugated anti-mouse F4/80; IL-10; TNF-α; IL-12/23; APC-conjugated anti-mouse F4/80; Tim-3; TNF-α; IL-10; Brilliant Violet 421-conjugated anti-mouse TGF-β1; CD206; TNF-α; IL-4; Brilliant Violet 510-conjugated anti-mouse CD86; TNF-α; CD4; Brilliant Violet 605-conjugated anti-mouse IL-17A; CD4 (Biolegend, San Diego, CA, USA). The HIF-2α antibody (Santa Cruz, CA, USA) was pretreated with the Lightning-Link APC Conjugation Kit (Innova Biosciences, Cambridge, UK). Before intracellular staining, a Fix/Perm kit (Biolegend, San Diego, CA, USA) was used to fix and permeabilize cells. Flow cytometry was carried out using a Beckman-Coulter CyAn ADP cytometer (Beckman-Coulter, Bria, CA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
The production of Angiopoietin-2, EGF, FGF, TGF-α, PDGF-AA, PDGF-BB, and VEGF by EVTs was evaluated using a Multi-Analyte Flow Assay Kit (Human Growth Factor Panel, Biolegend, San Diego, CA, USA).
The production of Angiopoietin-2, EGF, FGF, TGF-α, PDGF-AA, PDGF-BB, and VEGF by EVTs was evaluated using a Multi-Analyte Flow Assay Kit (Human Growth Factor Panel, Biolegend, San Diego, CA, USA).
3
Radiolabeling and Immune Profiling
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All chemical reagents, purchased from Meryer Chemical (China), Zhengzhou Alfa (China), J&K (China), Energy Chemical (China), Xi’an Ruixi Biological Technology Co., Ltd (China) and Macrocyclics, Inc. Cell counting kit-8 (CCK-8) was purchased from Biyuntian Biotechnology Institute. Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Yeasen Biotechnology (Shanghai) Co., Ltd. 89Zr was produced on an onsite cyclotron by the 89Y(p,n)89Zr reaction and purified to yield 89Zr(C2O4)2 solution. The cell Apoptosis 7-AAD Detection Kit was purchased from KeyGen Biotech Co., Ltd (Nanjing, China). FITC anti-mouse CD45 Antibody (#103107, 1:200), Pacific Blue™ anti-mouse/human CD11b Antibody (#101223, 1:100), APC/Cyanine7 anti-mouse F4/80 Antibody (#123117, 1:100), PE/Cyanine7 anti-mouse CD86 Antibody (#105013, 1:200), PE anti-mouse CD206 (MMR) Antibody (#141705, 1:100), PE-conjugated anti-mouse CD3 Antibody (#100205, 1:100), FITC-conjugated anti-mouse CD4 (#100406, 1:200), APC-conjugated anti-mouse CD8a Antibody (#100712, 1:200), Purified anti-HMGB1 Antibody (#651401, 1:100) were purchased from Biolegend. Mouse TNF-α, IL-6, IL-12p70 ELISA Kits and eFluor-450-conjugated anti-mouse Foxp3 antibody (#48-5773-82. 1:200) were obtained from Invitrogen. Alexa Fluor® 488 Anti-Calreticulin antibody [EPR3924] (ab196158, 1:100) was bought from Abcam.
4
Multiparametric Flow Cytometry of Lymphoid Cells
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For cell surface marker staining, draining lymph nodes (dLNs) of each mouse were individually collected and triturated into a single cell suspension. Then the cells were stained with different combinations of flow cytometry antibodies for 30 min at 4°C, which included APC-conjugated anti-mouse CD3 (eBioscience, USA), eFluor450-conjugated anti-mouse CD4 (eBioscience, USA), PE-conjugated anti-mouse CD8a (eBioscience, USA), APC-conjugated anti-mouse B220 (Biolegend, USA), Pacific Blue-conjugated anti-mouse GL-7 (Biolegend, USA), PE-conjugated anti-mouse CD95 (Biolegend, USA), FITC-conjugated anti-mouse CD4 (Biolegend, USA), PE-conjugated anti-mouse PD-1 (Biolegend, USA), and Brilliant Violet 421-conjugated anti-mouse CXCR5 (Biolegend, USA). After washing with staining buffer (eBioscience, USA), the cells were dispersed in 500 μL of staining buffer (eBioscience, USA) and analyzed by flow cytometry (Beckman Coulter, Cytoflex LX).
5
Mouse Lymphocyte Isolation and Phenotyping
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Mouse spleens were dissected out, ground, and filtered, and the filtrate was slowly added into 6 mL of mouse lymphocyte separation solution (Beijing Dakewe Biotechnology Co. Ltd., Beijing, China) along the wall of the centrifuge tube to isolate the mouse lymphocytes. The antibody system was configured with 100 μL of FACE solution (saline containing 2% fetal bovine serum) and 1 μL each of Brilliant Violet 510™-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD4, PerCP/Cyanine5.5-conjugated anti-mouse CD8a, APC-conjugated anti-mouse CD45 and PE-conjugated anti-mouse CD69 antibodies (BioLegend, San Diego, CA, USA) to every sample tube. After mixing, the cells were incubated at 4°C for 30 min in the dark. The excess antibodies were washed off with FACE solution, and the supernatant was discarded after centrifugation at 8000 rpm for 1 min. The sediment was dissolved with 200 μL of 4% paraformaldehyde in the dark for 20 min and the cells were detected by flow cytometry.
6
Multiparametric Immune Cell Profiling
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For analysis of surface molecules associated with CD4+ T cells, CD8+ T cells, regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and macrophages, single cell suspensions from spleens and tumors were stained with the following fluorescein-conjugated antibodies: FITC-conjugated anti-mouse CD4, PerCP-conjugated anti-mouse CD8a, FITC-conjugated anti-mouse Gr-1, Percp-conjugated anti-mouse CD11b and PE-conjugated anti-mouse F4/80 (all the above antibodies were from Biolegend, San Diego, CA, USA).
7
Flow Cytometric Analysis of Th1 and Th2 Cells
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Following treatment of purified CD4+ T cells with different drugs, the concentrations of Th1 cells and Th2 cells were investigated by flow cytometry (BD Bioscience, USA). To facilitate the intracellular staining of T helper cells, all cells were stimulated with Cell Activation Cocktail (with Brefeldin A) (BioLegend, USA) for 5 hours before detection. Staining of T helper cells for flow cytometry requires two steps: extracellular staining and intracellular staining. After the harvested cells were washed with FACS buffer, FITC-conjugated anti-mouse CD4 (BioLegend, USA) was added to the cells for 30 minutes at RT. To decrease the nonspecific Fc receptor-mediated Ab staining, cells were preincubated with mouse IgG for 20 minutes at 4°C. Then, the FIX&PERM Kit (FMS, China) was used according to the manufacturer's instructions to fix and permeabilize the cells. APC-conjugated anti-mouse IFN-γ (BioLegend, USA) and PE-conjugated anti-mouse IL-4 (BioLegend, USA) were added to bind to intracellular cytokines. Th1 cells and Th2 cells were labeled with CD4 + IFN-γ and CD4 + IL-4, respectively.
8
Multiparametric Flow Cytometry Analysis
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Single-cell suspensions were prepared from the peripheral blood of mice using standard procedures. Following red blood cell lysis, Fc receptors were blocked with anti-CD16/32 Ab (2.4G2), and cells were incubated with antibody in 0.5% FBS in PBS for 30 min, and then washed in FACS buffer before analysis. Data were collected on a FACSCanto™ II (BD Biosciences San Jose, CA, USA) instrument and analyzed using FlowJo software (Tree Star, Ashland, OR, USA). The Antibodies used for staining are as follows. FITC-conjugated anti-mouse CD4 (GK1.5), NKp46 (29A1.4), Siglec-F (S17007L), F4/80 (BM8), PE-conjugated anti-mouse T cell receptor γ/δ (GL3), CXCR5 (L138D7), α-GalCer : CD1d complex (L363), Gr-1 (RB6-8C5), 317 (927), PE/Cy7-conjugated anti-mouse CD8a (53-6.7), CD25 (3C7), B220 (RA3-6B2), CD11b (M1/70), CD11c (N418), APC-conjugated anti-mouse Slamf1 (W19132B) were purchased from BioLegend (San Diego, CA, USA).
9
Isolation and Characterization of Spinal Cord Immune Cells
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Leukocytes were isolated from spinal cords and cervical lymph nodes using a Percoll gradient method [16 (link)–18 (link)]. Briefly, tissues were dissociated by grinding and passed through a nylon strainer. Cells were centrifuged with 80% and 40% Percoll at 1300×g at room temperature. Cells at the interface between 40 and 80% Percoll were taken. For intracellular staining, isolated cells were stimulated with PMA (Sigma, 50 ng/mL) and ionomycin (Sigma, 1 μg/mL) along with brefeldin A (Golgi Plug, Becton-Dickenson) for 4 h and were fixed with cell fixation/permeabilization solution (BD Cytofix/Cytopermtm) according to manufacturer’s protocol. Antibodies used for flow cytometry were as follows: FITC conjugated anti-mouse CD4 (Biolegend), PerCP conjugated anti-mouse CD8a, APC-conjugated anti-mouse IFNγ, and PE-conjugated anti-mouse IL-17 (eBioscience).
10
Multicolor flow cytometry analysis of immune cell subsets
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For T lymphocyte subpopulations assay, single-cell suspension of splenocytes and mesenteric lymph nodes was adjusted to a concentration of 1 × 106 cells/mL. T-cell specific markers were stained using PerCP-Cy5.5-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD4, Pacific Blue-conjugated anti-mouse CD8a, PE-conjugated anti-mouse CD25, PE/Cy7-conjugated anti-mouse NK-1.1 (only in splenocyte) and Alexa Fluor 647-conjugated anti-mouse foxP3 (only in mesenteric lymph node lymphocytes) (BioLegend, San Diego, CA, USA). We then conducted fluorescence activated cell sorting (FACS) and data analysis using a Beckman Gallios™ flow cytometer (Beckman Coulter, Pasadena, CA, USA), after adjusting the instrument settings using the control cells stained with isotype-matched antibodies.
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