The Kupffer cells and MDMs isolated using magnetic bead separation were washed and resuspended in FACS buffer (PBS, 1% FBS). The cells were then incubated with Fc blocking buffer (BD Biosciences; diluted 1:20) for 10 mins at 4°C. The cells were then rinsed and centrifuged at 300 g for 5 minutes. The cell pellet was then resuspended with anti-F4/80 Alexa Fluor-488 and anti-CD11b APC/Cy7 (BioLegend, San Diego, CA) and incubated for 30 minutes at 4°C. The cells were then washed twice and fixed in formalin (Sigma) for 15 minutes. After cells were fixed, they were washed twice and resuspended in FACs buffer. Fluorescence was then detected using an Attune NxT flow cytometer (Life Technologies, Carlsbad, CA) and the data were analyzed using Attune NxT software.
Fc blocking buffer
Manufactured by BD
Fc blocking buffer is a solution used to prevent non-specific binding of antibodies to Fc receptors in immunoassays. It helps to reduce background signal and improve the specificity of the assay.
Automatically generated - may contain errors
Lab products found in correlation
Formalin, by Merck Group (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Anti f4 80 alexa fluor 488 and anti cd11b apc cy7, by BioLegend (2 mentions)
Brilliant stain buffer, by BD (2 mentions)
Ter119, by BioLegend (1 mentions)
Cd11b, by Cytek Biosciences (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Cd200, by BD (1 mentions)
Cd105, by BioLegend (1 mentions)
Aria 2, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (1 mentions)
5 protocols using fc blocking buffer
1
Isolation and Flow Cytometry Analysis of Kupffer Cells and MDMs
2
Mouse Bone Marrow Cell Immunophenotyping
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Bone marrow cells were flushed from the femurs and tibias of 2-month-old mice (C57BL/6J) and incubated for 2 min at room temperature with BD Pharm Lyse hypotonic lysis buffer (BioLegend, 420301) for red blood cell (RBC) lysis. Cells were washed twice with cold fluorescence-activated cell sorting (FACS) buffer, incubated with Fc blocking buffer (BD Biosciences, 564765) for 15 min at 4°C, and treated with antibody cocktail including CD11b (Tonbo Biosciences, 20-0112), CD45R/B220 (Tonbo Biosciences, 65-0452), CD117 (Tonbo Biosciences, 60-1172), CD3 (Tonbo Biosciences, 50-0031), Ter119 (BioLegend, 116233), and Ly6C (BioLegend, 128017) in cold FACS buffer. After treatment with DAPI, cells were subjected to FACS analysis using a BD LSR II flow cytometer (BD Biosciences). The data were analyzed using FlowJo (v.10.1).
3
Isolation of Skeletal Progenitors via FACS
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Human BMA purchased from StemExpress (BMEDT010F) was incubated for 10 min at room temperature with BD Pharm Lyse hypotonic lysis buffer (BD Biosciences, 555899) for RBC lysis. Cells were washed with cold FACS buffer twice, incubated with Fc blocking buffer (BD biosciences, 564765) for 15 min at 4 °C, and treated with antibody cocktail including CD31 (1:100, BD biosciences, 560984), CD45 (1:100, BD biosciences, 560976), CD235a (1:100, BD biosciences, 561017), CD90 (1:100, BD biosciences, 566219), CD200 (1:100, BD biosciences, 564114), CD105 (1:100, BioLegend, 323217) in brilliant stain buffer (BD biosciences, 563794). After treatment with DAPI, cells were subjected for FACS analysis using a Becton Dickinson Aria II equipped with five lasers (BD biosciences). The data were analyzed using FlowJo (v.10.1). The strategy to sort skeletal progenitors is diagrammed in Supplementary Fig. 3 .
4
Isolation and Flow Cytometry Analysis of Kupffer Cells and MDMs
5
Single-Cell Immunophenotyping by Flow Cytometry
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Prepared single-cell suspensions were washed twice with ice-cold FACS buffer (2% serum + 1 mM EDTA in PBS) and incubated with Fc blocking buffer (1:100 dilution; BD bioscience, cat. 553142 for mouse and cat. 564765 for human) for 15 min at 4 °C. Primary antibody dilutions were prepared in Brilliant Stain Buffer (BD bioscience, cat. 563794). Cells were incubated in the dark for 30 min at 4 °C with primary antibody solution and washed 2 times with FACS buffer. When secondary antibodies were used, cells were further incubated with secondary antibody solution for 20 min. Cells were then washed two times and re-suspended in FACS buffer with DAPI (BD bioscience, cat. 564907). FACS was performed using a Becton Dickinson Aria II equipped with 5 lasers (BD bioscience). Beads (Invitrogen, cat. 01-3333-42) were used to set initial compensation. Fluorescence minus one (FMO) controls were used for additional compensation and to assess background levels for each stain. Gates were drawn as determined by internal FMO controls to separate positive and negative populations for each cell surface marker. Typically, 2.5 million events were recorded for each FACS analysis, and the data were analyzed using FlowJo (v10.8.1).
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