Single cells were obtained from lungs digested by collagenase. The cells were stained for 1 h with abs PE Rat Anti-Mouse F4/80 (BD Pharmingen Cat# 565410), PE/Cy7 Anti-Mouse/Human CD11b (BioLegend Cat# 101215), PerCP/Cyanine5.5 Anti-Mouse CD45 (BioLegend Cat# 103132), and FITC Anti-Mouse Ly-6G/Ly-6C (Gr-1) (BioLegend Cat# 108406) diluted in PBS at a 1: 1,000. For compensation, single stained samples were set. Cells were analyzed on BD FACSymphony (BD). Data were generated using FlowJo V10 (Treestar, Stanford, CA).
Pe rat anti mouse f4 80
Manufactured by BD
Sourced in United States
The PE Rat Anti-Mouse F4/80 is a laboratory reagent used for the detection and identification of F4/80 antigen, which is a specific marker for mouse macrophages. This reagent is conjugated with the fluorescent dye Phycoerythrin (PE) and can be used in flow cytometry and other immunoassay applications.
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Lab products found in correlation
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Fitc hamster anti mouse cd3e, by BD (2 mentions)
Apc cy7 rat anti mouse cd45, by BD (2 mentions)
Apc anti mouse cd11c, by BioLegend (2 mentions)
R1010, by Solarbio (2 mentions)
Pe cy7 anti mouse human cd11b, by BioLegend (1 mentions)
Percp cyanine5.5 anti mouse cd45, by BioLegend (1 mentions)
Fitc anti mouse ly 6g ly 6c gr 1, by BioLegend (1 mentions)
Facsymphony, by BD (1 mentions)
Pe cy7 rat anti mouse cd8a, by BD (1 mentions)
Pharmingen staining buffer, by BD (1 mentions)
Fitc rat anti cd11b, by BD (1 mentions)
Fc block, by BD (1 mentions)
Facscelesta, by BD (1 mentions)
Facsaria 2 instrument, by BD (1 mentions)
Rat anti mouse cd206, by BD (1 mentions)
Fitc rat anti mouse cd11b, by BD (1 mentions)
Apc rat anti mouse cd11b, by BD (1 mentions)
Fitc rat anti mouse cd8a, by BD (1 mentions)
Apc mouse anti human hla dr, by BD (1 mentions)
7 protocols using pe rat anti mouse f4 80
1
Lung cell flow cytometry analysis
2
Flow Cytometry Analysis of Mouse Spleen Cells
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The FCA samples of mouse spleen were prepared as previously reported [16 (link)]. In brief, mouse spleen tissues were ground in PBS and then passed through a 70 μm filter. Spleen samples were then incubated in red blood cell lysis buffer (R1010, Solarbio, Beijing, China) for 20 min to lyse red blood cells, followed by resuspension of the spleen sample with PBS. The prepared single-cell suspension from spleen was stained with FVS (564997, BD Pharmigen, San Diego, CA, USA), APC-Cy™7 Rat anti-mouse CD45 (557659, BD), PE Rat anti-mouse F4/80 (565410, BD), APC anti-mouse CD11c (117310, Biolegend, San Diego, CA, USA), FITC Hamster anti-mouse CD3e (553061, BD), PerCP/Cyanine5.5 anti-mouse CD4 (100434, Biolegend) and PE-Cy™7 Rat anti-mouse CD8a (552887, BD) for 30 min. After washing with PBS, the stained cells were analyzed using a flow cytometer (BD Bioscience, San Diego, CA, USA).
3
Multiparameter Immunophenotyping of Macrophage Subsets
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After different treatments, RAW264.7 cells and BMDM cells were resuspended in BD Pharmingen staining buffer (cat# 554657; BD Biosciences). Cells were incubated with FC block (cat# 553141; BD Biosciences) for 20 minutes. Subsequently, cells were washed and resuspended in 100 μl of BD Pharmingen staining buffer and incubated with PE Rat Anti-Mouse F4/80 (cat# 565410; BD Biosciences), FITC Rat Anti-CD11b (cat# 557396; BD Biosciences), PE-CyTM7 Rat Anti-Mouse CD86 (cat# 560582; BD Biosciences), Alexa Flour® 647 Rat Anti-Mouse CD206 (cat# 565250; BD Biosciences) on ice for 30 minutes. Cells were washed three times with staining buffer and resuspended in staining buffer. Cells were centrifuged and resuspended in staining buffer for FACS analysis (FACS Celesta; BD Bioscience, San Jose, USA). FACS data analysis was performed using FLOWJOTM Software.
4
Flow Cytometry Analysis of Macrophage Markers
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The RAW264.7 cells were harvested from a 6-well plate and washed with cold PBS three times. Then, the cells were stained with PE Rat Anti-Mouse F4/80 (565410, BD, United States), FITC Rat Anti-Mouse CD11b (557396, BD, United States), APC-Cy7 Hamster Anti-Mouse CD11c (561241, BD, United States), PE-Cy7 Rat Anti-Mouse CD16/CD32 (560829, BD, United States) and Rat Anti-Mouse CD206 (565250, BD, United States) antibodies at 4°C for 30 min. After staining, the cells were washed with PBS three times and resuspended with 100 μL of PBS for FACS analysis using a BD FACSAria II instrument (BD, United States). The data were further analyzed using Flowjo software 7.6.
5
Immune Cell Phenotyping from Tissues
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Immune cells from animal tissue or blood were separated by using HISTOPAQUE®‐1077(10771, Sigma), and then the dissociated lymphocytes were resuspended in PBS and stained for surface markers for 20 minutes with antibodies: PerCP‐CyTM 5.5 rat anti‐mouse CD45 (550994, BD Biosciences, 1:200), FITC rat anti‐mouse CD8a (553030, BD Biosciences, 1:500), APC rat anti‐mouse CD11b (553312, BD Biosciences, 1:200), PE rat anti‐mouse F4/80 (565410, BD Biosciences, 1:200), APC anti‐mouse CD206 (MMR) (141707, BD Biosciences, 1:200), APC mouse anti‐human HLA‐ABC (555555, BD Biosciences, 1:200), APC mouse anti‐human HLA‐DR (560896, BD Biosciences, 1:125), and BV421 rat anti‐mouse CD119 (740032, BD Biosciences, 1:200). Flow cytometry data were obtained on an LSRFortessa, and data analysis was achieved through FlowJo software (Tree Star).
6
Immune Response to Extracellular Vesicles
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All animal experiments were approved by the Animal Care and Use Committee of West China Hospital, Sichuan University (permit no. 20230330008) and were performed according to the guidelines of the National Institutes of Health (NIH). BALB/c mice (male, 8 weeks) were purchased from Byrness Weil Biotech Ltd. (Chengdu, China) and housed in a pathogen-free facility. Normal mice were randomly divided into three groups (n = 5) and intravenously injected with 100 μl of PBS, EVGLN− preparations (30 μg each mouse), or EVGLN+ preparations (30 μg each mouse). At 4 hours after injection, the mice were anaesthetized, and the spleens were collected and prepared as single-cell suspensions to detect immune cells. In brief, mouse spleen tissues were ground in PBS and then passed through a 70-μm filter. The red blood cells in spleen samples were removed using a Mouse Erythrocyte Lysing Kit (Solarbio, Beijing, China). The single-cell suspension was stained with FVS (564997, BD), APC-Cy7 rat anti-mouse CD45 (557659, BD Pharmingen), PE rat anti-mouse F4/80 (565410, BD), APC-conjugated anti-mouse Ly6G (560599, BD), and BV421-conjugated anti-mouse Ly6C (562727, BD) for 30 min. After washing with PBS, the stained cells were analyzed using a flow cytometer (LSRFortessa).
7
Immunophenotyping of Mouse Splenic Leukocytes
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The FCA samples of mouse spleen were prepared as previously reported [19] . In brief, mouse spleen tissues were ground in PBS and then passed through a 70 μm filter.
Spleen samples were then incubated in red blood cell lysis buffer (R1010, Solarbio, Beijing, China) for 20 min to lyse red blood cell, followed by re-suspending the spleen sample with PBS. The prepared single-cell suspension from spleen was stained with FVS (564997, BD Pharmigen, San Diego, CA, USA), APC-Cy TM 7 Rat anti-mouse CD45 (557659, BD), PE Rat anti-mouse F4/80 (565410, BD), APC anti-mouse CD11c (117310, Biolegend, San Diego, CA, USA), FITC Hamster anti-mouse CD3e (553061, BD), PerCP/Cyanine5.5 anti-mouse CD4 (100434, Biolegend) and PE-Cy TM 7 Rat antimouse CD8a (552887, BD) for 30 min. After washing with PBS, the stained cells were analyzed using a flow cytometer (BD Bioscience, San Diego, CA, USA).
Spleen samples were then incubated in red blood cell lysis buffer (R1010, Solarbio, Beijing, China) for 20 min to lyse red blood cell, followed by re-suspending the spleen sample with PBS. The prepared single-cell suspension from spleen was stained with FVS (564997, BD Pharmigen, San Diego, CA, USA), APC-Cy TM 7 Rat anti-mouse CD45 (557659, BD), PE Rat anti-mouse F4/80 (565410, BD), APC anti-mouse CD11c (117310, Biolegend, San Diego, CA, USA), FITC Hamster anti-mouse CD3e (553061, BD), PerCP/Cyanine5.5 anti-mouse CD4 (100434, Biolegend) and PE-Cy TM 7 Rat antimouse CD8a (552887, BD) for 30 min. After washing with PBS, the stained cells were analyzed using a flow cytometer (BD Bioscience, San Diego, CA, USA).
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