For classification of granulocytes, cells from BALF were stained with Zombie Aqua™ solution for 10 min in the dark, stained for 30 min with antibodies for markers as follows: FITC Rat Anti-Mouse CD45 (#553079, BD, USA), PE/Cyanine7 anti-mouse CD11c (#117317, BioLegend, USA), BB700 Rat Anti-Mouse CD11b (#566416, BD, USA), APC Rat Anti-Mouse Ly-6G (#560599, BD, USA), PE Rat Anti-Mouse Siglec-F (#552126, BD, USA), BV421 Rat Anti-Mouse F4/80 (#565411, BD, USA). Absolute cell counts were calculated on the basis of Precision Count BeadsTM (#424902, BioLegend, USA). Data were collected on a BD Biosciences FACSVerse Flow Cytometer and analyzed using FlowJo software. The Flow cytometry gating strategy is shown in Supplementary Fig. S1F .
Bv421 rat anti mouse f4 80
Manufactured by BD
Sourced in United States
The BV421 Rat Anti-Mouse F4/80 is a monoclonal antibody that targets the F4/80 antigen, a glycoprotein expressed on the surface of mouse macrophages. This antibody is conjugated with the BV421 fluorescent dye, which is useful for flow cytometry and other immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsverse flow cytometer, by BD (1 mentions)
Precision count beads, by BioLegend (1 mentions)
Fitc rat anti mouse cd45, by BD (1 mentions)
Pe rat anti mouse siglec f, by BD (1 mentions)
Pe cyanine7 anti mouse cd11c, by BioLegend (1 mentions)
Apc rat anti mouse ly 6g, by BD (1 mentions)
Attune nxt, by Thermo Fisher Scientific (1 mentions)
Apc rat anti mouse cd11b, by BD (1 mentions)
Collagenase p, by Boehringer Ingelheim (1 mentions)
Hank s solution, by Beyotime (1 mentions)
Pe hamster anti mouse cd11c, by BD (1 mentions)
Fitc rat anti cd11b, by BD (1 mentions)
Pe rat anti mouse il 17a, by BD (1 mentions)
Fitc hamster anti mouse cd3e, by BD (1 mentions)
Transcription factor buffer set, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Pe cy7 rat anti mouse cd4, by BD (1 mentions)
Fixable viability stain 780, by BD (1 mentions)
Alexa fluor 647 rat anti mouse cd206, by BD (1 mentions)
Alexa fluor 647 rat anti mouse foxp3, by BD (1 mentions)
5 protocols using bv421 rat anti mouse f4 80
1
Granulocyte Identification in BALF
2
Tissue Digestion and FACS Immunophenotyping
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Freshly harvested pancreatic and colonic tissues were digested in 1.0 mg/mL collagenase-P (Boehringer, Mannheim, Germany) solution at 37°C for 15 min and filtered through 75 µm filters with hank’s solution (Beyotime, Shanghai, China). Single-cell suspensions were incubated for 30 min at 4°C in hank’s solution with the following mAbs: APC Rat Anti-Mouse CD11b, BV421 Rat Anti-Mouse F4/80, Alexa Fluor 700 Rat Anti-Mouse Ly-6G, PE Hamster Anti-Mouse CD11c, FITC Rat Anti-Mouse MHCII, and PerCP-Cy5.5 Rat Anti-Mouse CD8a (BD Pharmingen, CA, USA). Gating method of fluorescence-activated cell sorting was programmed as CD11b+ Ly-6G+ (for neutrophils), CD11b+ F4/80+ (for macrophages), CD11c+ MHCII+ (for conventional dendritic cells, cDCs), and CD8a+CD11c+ MHCII+ (for plasmacytoid dendritic cells, pDCs). Flow cytometer was performed on Attune NxT (Thermo Fisher Scientific, MA, USA). Data were analyzed using ACEA NovoExpress software (Novo Express International, Inc., South San Francisco, CA, USA).
3
Comprehensive Immune Cell Profiling by Flow Cytometry
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Flow cytometry was performed on a CytoFLEX (Beckman), and data were analyzed by FlowJo software. The live cells were observed by the Fixable Viability Stain 780 (Cat#565388, BD Pharmingen). For cell-surface staining, cells were stained with BV510 Rat Anti-Mouse CD45(Cat#563891, BD Pharmingen), FITC Hamster Anti-Mouse CD3e (Cat#553061, BD Pharmingen), PE-Cy7 Rat Anti-Mouse CD4 (Cat#552775, BD Pharmingen), BV421 Rat Anti-Mouse CD25 (Cat#562606, BD Pharmingen), FITC Rat Anti-CD11b (Cat#557396, BD Pharmingen), BV421 Rat Anti-Mouse F4/80 (Cat#565411, BD Pharmingen), PE Rat Anti-Mouse CD86(Cat#561963 BD Pharmingen), Alexa Fluor 647 Rat Anti-Mouse CD206 (Cat#565250 BD Pharmingen). For intracellular staining, cells were stained with PE Rat Anti-Mouse IL-17A (Cat#561020 BD Pharmingen), Alexa Fluor 647 Rat anti-Mouse Foxp3(Cat#560402 BD Pharmingen). According to the reagent vendor’s instructions, Purified Rat Anti-Mouse CD16/CD32 is used to exclude background fluorescent signal interference, Cells fixation and permeabilization Fixation/Permeablization Kit (Cat#554714 BD Pharmingen) and Transcription Factor Buffer Set (Cat#562574 BD Pharmingen) were used for cell fixation and permeabilization.
4
Cardiac Immune Cell Isolation and Phenotyping
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Mouse heart (left ventricle) was perfused107 , excised, minced, digested with Collagenase II (#LS004174, Worthington Biochemical Corporation) and Dispase II (#494207800, Roche). LV tissue mixture was then mechanically disrupted and filtered through 70-μm cell strainer to obtain single-cell suspensions. After blocking with CD16/32 (#14-0161-85, Thermo Fisher Scientific, 1:100) at RT for 10 min, cells were collected by centrifugation, subjected to live-death dye and surface antibody staining, and analyzed or sorted by flow cytometry using BD FACS ARIA system, and the data were analyzed using FlowJo v10. Cardiac macrophages, cardiac monocytes, or Ly6Chigh monocytes were gated from whole heart cells as CD45+ CD64+ CD11b+ F4/80+ Ly6G− or CD45+ CD64+ CD11b+ or CD45+ CD64+ CD11b+ Ly6Chigh cells, respectively. The specific antibodies used for flow cytometry are listed as follows: Anti-Ly-6C Monoclonal Antibody PerCP-Cyanine5.5 (#45-5932-82, Thermo Fisher Scientific, 1:25), APC Rat anti-mouse Anti-CD11b (#561690, BD Biosciences, 1:100), FITC Mouse Anti-Mouse CD45.2 (#561874, BD Biosciences, 1:100), APC-H7 Rat Anti-Mouse Ly-6G (#565369, BD Biosciences, 1:200), PE Mouse anti-Mouse CD64 (#558455, BD Biosciences, 1:100), BV421 Rat Anti-Mouse F4/80 (#565411, BD Biosciences, 1:100), mouse anti-mouse CD45 (#561874, BD Biosciences, 1:100), rat anti-mouse IRF7-PE (#12-5829-82, Biolegend, 1:100).
5
Analysis of Peritoneal Macrophages
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Macrophages collected from peritoneal lavage were labeled with BV421 Rat Anti-Mouse F4/80 (BD PharmingenTM, San Jose, CA, United States) at 4°C for 15 min. For measuring cell surface expression of TLR4, macrophages were stained with PE conjugated anti-mouse CD284 (TLR4) antibody (eBioscience affymetrix, San Diego, CA, United States) for 30 min. For detecting cell death, cells were incubated with PI and Annexin V (BD PharmingenTM, San Jose, CA, United States) for 15 min at room temp, and the double-stained cells were counted as dead cells. A total of 20,000 events were collected and the cells were analyzed using FACScalibur cytometer (BD Biosciences, San Jose, CA, United States). Peritoneal macrophages were identified as F4/80high. The final data were analyzed by FlowJo-V10 software (Tree Star, Ashland, OR, United States).
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