Flow cytometry was performed on leukocytes isolated from organs as described above. Cells were washed in FACS buffer containing HBSS, 1% BSA, 4.17mM sodium bicarbonate, and 3.08 mM sodium azide and incubated for 15 minutes at room temperature with Fc Block (anti-CD16/32; Tonbo, San Diego, CA) except for stains for FcγRIIb. Cells were then incubated for 30 minutes at 4°C with the following antibodies: anti-CD11c-FITC (Tonbo), anti-CD11b-V450 (Tonbo), anti-CD45.2-APCCy7 (Tonbo), anti-CD64-PE (BD, San Jose, CA), anti-CD36-APC (BD), anti-MHC Class II-PerCP-Cyanine5.5 (Tonbo), anti-CD32b-PE (Thermofisher, Waltham, MA), anti-TCRβ-V450 (Tonbo), and anti-CD4-PeCy7 (Tonbo). All samples were washed, and either fixed in 2% paraformaldehyde (PFA) or permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA) for intracellular staining overnight at 4°C. Cells were then stained an additional 30 minutes with anti-FoxP3-FITC (eBioscience) followed by washing and fixation in 2% PFA. Samples were run using a MACSQuant Analyzer (Miltenyi, Auburn, CA) and analyzed using FlowJo software.
Fc block anti cd16 32
Manufactured by Cytek Biosciences
Fc Block (anti-CD16/32) is a laboratory reagent used to block non-specific Fc receptor binding in flow cytometry experiments. It contains antibodies that bind to the CD16 and CD32 receptors, preventing them from interfering with the primary antibody-antigen interactions being analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Diva acquisition software, by BD (2 mentions)
Anti foxp3 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd11c fitc, by Cytek Biosciences (1 mentions)
Anti cd64 pe, by BD (1 mentions)
Anti cd36 apc, by BD (1 mentions)
Anti cd4 pecy7, by Cytek Biosciences (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Percp cy5, by BioLegend (1 mentions)
Apc fire 750, by BioLegend (1 mentions)
Facs lsrfortessa, by BD (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Alexaflour 700, by BioLegend (1 mentions)
Zombie nir, by BioLegend (1 mentions)
4 protocols using fc block anti cd16 32
1
Multicolor Flow Cytometry for Leukocyte Analysis
2
Multiparameter Flow Cytometry of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 1 × 106 mononuclear cells suspended in PBS were stained using incubation with 1 µg Fc Block (anti-CD16/32, Tonbo Biosciences) for 15 min at 4 °C. Cells were then stained with the following antibodies: CD45 (APC/Fire 750-Biolegend Clone: 13/2.3), CD11c (APC-Biolegend, Clone: N418) MHCII (PerCp Cy5.5-Biolegend, Clone: M5/114.15.2), CD88 (PE, Biolegend, Clone: 20/70), CD317 (BV421, Biolegend, Clone: 927), CD45.1 (FITC-Biolegend Clone: A20), and CD45.2 (BV605, Biolegend, Clone: 104) for 45 min at 4 °C. Events were recorded via BD FACS LSR Fortessa (The Moody Foundation Flow Cytometry Facility, UT Southwestern), equipped with Diva acquisition software (BD Bioscience). For FACS sorting, BD FACSARIA-II SORP (The Moody Foundation Flow Cytometry Facility, UT Southwestern) was utilized. Cells were gated according to morphology based on their side scatter (SSC) vs. forward scatter (FSC) distributions. Doublets were excluded (FSC-A vs. FSC-H and SSC-A vs. SSC-H, where A corresponds to area and H corresponds to height). Gating for FACS sorting consisted of singlet CD45+MHCII+CD88+CD317+ CD45.1+ cells or CD45+MHCII+CD88+CD317+CD45.2+ cells. In each sample, a minimum of 50 × 103 live events were recorded. FlowJo software (BD Bioscience) was used for data analysis.
3
Flow Cytometry Cell Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Staining of cells was performed using a standard protocol. In brief, cells were counted manually by hemocytometer with trypan blue exclusion and dispensed into flow cytometry tubes, centrifuged, and resuspended in staining buffer containing Fc block (anti-CD16/32, 10 μg/ml, Tonbo Biosciences, San Diego, CA) for 25-minute on ice. Antibody cocktail (S3 Table ) was added to surface stain cells for 30 minutes on ice, followed by washing in stain buffer. Viability was determined using live/dead aqua according to manufacturer’s instructions (ThermoFisher, Waltham MA). All flow cytometry data was acquired on a LSRII (BD Biosciences, Franklin Lakes NJ) using DIVA software, with analysis using FlowJo (Treestar, Eugene OR)
4
Comprehensive Immune Cell Phenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 1 x 10 6 mononuclear cells suspended in PBS were stained using fixable viability dye (Zombie NIR-Biolegend), and incubated for 15 minutes at room temperature following manufacturer's recommendation. Cells were then washed using 2% FBS-PBS (FACs buffer) at 1500 rpm for 5 minutes, followed by incubation with 1 µg Fc Block (anti-CD16/32, Tonbo Biosciences) for 15 minutes at 4ºC. Cells were then stained with the following antibodies: CD45 (Alexa Flour 700-Biolegend, Clone: 30-F11), CD11b (V450-BD Horizon, Clone: M1/70), CD19 (BV510-Biolegend, Clone: MHCII (BV785, Biolegend, Clone: M5/114.15.2), CD49d (α4-integrin; FITC, Biolegend, Clone: R1-2), CD88 (PE, Bilegend, Clone: 20/70), CD317 (BV421, Biolegend, Clone: 927) CD26 (FITC, Biolegend, Clone: H194-112) for 45 minutes at 4ºC. Events were recorded via BD FACS LSR Fortessa (The Moody Foundation Flow Cytometry Facility, UT Southwestern), equipped with Diva acquisition software (BD Bioscience). Cells were gated according to morphology side scatter (SSC-A) vs forward scatter (FSC-A). Doublets were excluded (FSC-A vs FSC-H and SSC-A vs SSC-H). Live cells were selected using the viability dye. The complete gating strategy is presented in Supplementary Figure 1. In each sample a minimum of 50 x 10 3 live events were recorded. FlowJo software (BD Bioscience) was used for data analysis.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!