Isolated cells were incubated with rat anti-mouse CD16/CD32 (eBioscience, San Diego, CA, USA) for 15 min at 4°C. The cells were then incubated with primary antibody cocktails for 30 min at 4°C in the dark (for CACs, Alexa700 anti-mouse CD45, Cat# 560510, BD Biosciences, San Jose, CA, USA; BV650 anti-mouse Flk1, Cat# 740539, BD Biosciences; PerCP-eFluor710 anti-mouse CD31, Cat# 46–0311-82, eBioscience). Immune cells and monocytes were identified after staining with the following antibodies: PerCPCy5.5 anti-mouse NK 1.1, Cat# 551114, BD Biosciences; BV500 anti-mouse CD3, Cat# 560771, BD Biosciences; PE/Cy7 anti-mouse CD11c, Cat# 558079, BD Biosciences; PE anti-mouse Ly6G, Cat# 551461, BD Biosciences; BV421 anti-mouse CD115, Cat# 135513, Biolegend, San Diego, CA, USA; FITC anti-mouse Ly6C, Cat# 128006, Biolegend). After washing, the cells were then stained with flexible viability dye eFluor 780 (Cat# 65–0865-18, eBioscience) for 30 min at 4°C, washed with PBS twice, then fixed with 1% paraformaldehyde (PFA) for flow cytometry.
Rat anti mouse cd16 cd32
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Rat anti-mouse CD16/CD32 is a monoclonal antibody that binds to the mouse CD16 and CD32 receptors. CD16 and CD32 are Fc gamma receptors expressed on various immune cells, including macrophages, dendritic cells, and natural killer cells. This antibody can be used to block Fc receptor-mediated binding and activation of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
7 aad viability dye, by Thermo Fisher Scientific (2 mentions)
Rat serum, by STEMCELL (2 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Anti mouse ly6g pe, by BD (1 mentions)
Percpcy5 5 anti mouse nk 1, by BD (1 mentions)
Fitc anti mouse ly6c, by BioLegend (1 mentions)
Collagenase 1, by Solarbio (1 mentions)
Hyaluronidase, by Solarbio (1 mentions)
Fortessa x20, by BD (1 mentions)
Aria 2, by BD (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Fixation buffer, by BD (1 mentions)
Fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Streptavidin pe, by Bio-Rad (1 mentions)
Apc cd11c, by Bio-Rad (1 mentions)
Fitc streptavidin, by Bio-Rad (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Anti mouse rat foxp3 staining set, by Thermo Fisher Scientific (1 mentions)
8 protocols using rat anti mouse cd16 cd32
1
Multicolor Flow Cytometry of Immune Cells
2
Staining and Sorting Murine ILC2 Progenitors
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Cells were first blocked with purified rat anti-mouse CD16/CD32 (2 μg/ml) (eBioscience) and rat serum (20 μg/ml) (Stem Cell). Cells were then stained with specific antibodies of interest in the FACS buffer (Supplementary Table 1
3
Isolation and Identification of Mouse Endothelial and Leukocyte Cells
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MHECs and mouse peripheral blood leukocytes were isolated from sAC-C2flox/flox and sAC-C2flox/flox, VE-Cre+ mice as described above. For both cell types, RBCs were lysed for 15 min at room temperature, washed twice, and then incubated with rat anti–mouse CD16/CD32 (eBioscience) for 15 min at room temperature to block Fc receptors and washed twice more. Heart tissue homogenate was then labeled with FITC rat anti–mouse CD31 and APC rat anti–mouse CD45, or isotype control, for 30 min at 4°C. Mouse leukocytes were labeled with PE rat anti–mouse Ly6G (Gr1) and FITC rat anti–mouse CD31, or isotype control. Cells were then sorted at the Northwestern University Robert H. Lurie Comprehensive Cancer Center Flow Cytometry Core Facility using a FACSAria SORP 4-laser cell sorter (BD). MHEC were deemed those FITC CD31+/APC CD45− in the heart tissue homogenate. Leukocytes were deemed those PE Ly6G+/APC CD45+ in the peripheral blood.
4
Quantifying Renal CD133+ Cell Percentage
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The percentage of CD133+ cells in mouse renal tissue is expressed by the proportion of CD133+ cells that occupy the total number of nucleated cells in the renal tissue. The right kidney of the mice was obtained at the indicated time point of the experiment, and the percentage of CD133+ cells occupying the total renal cells in each group was determined. The experimental procedure is as follows: The kidneys collected from each group were washed with 10% heparin in 1x PBS to remove the blood, then were cut into fragments in 1x PBS at 4 °C. Then hyaluronidase (1 mg/ml), DNA lyase (1 mg/ml), collagenase I (150 units/ml) (Solarbio, Beijing, China) were added and the samples were kept at 37 °C in water bath for 1 hour with a gentle vertex in every 15 mins. The undigested tissue was filtered out by using 70 µm nylon cell filter (ThermoFisher, USA). The flow-through mixture was centrifuged at 600 g for 10 min at 4 °C, and the supernatant was removed. The cells were washed again by 1x PBS, and then the purified Rat anti-mouse CD16/CD32 (eBioscience, USA) were added as Fc blocking purposes. Followed by addition 30 minutes of anti-mouse CD133 staining (Biolegend, USA) and analyzed by flow cytometry (BD Biosciences, USA). Each sample counted at least 1000000 cells.
5
Multidimensional Flow Cytometry Analysis
6
Immunophenotyping of ILC2 Progenitors
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Cells were first blocked with purified rat anti-mouse CD16/CD32 (2 μg/ml) (eBioscience) and rat serum (20 μg/ml) (Stem Cell) to prevent non-specific binding of antibodies. Cells were then stained with specific antibodies of interest in the FACS buffer (2% FCS, 1 mM EDTA, and 0.05% azide in PBS). For BM-derived ILC2Ps FACS sorted for culture, the cells were stained in ILC media. Viable cells were identified using the viability dye 7-AAD (eBioscience). The samples were either resuspended in the FACS buffer for acquisition, or fixed overnight at 4 o C for intracellular staining the next day using a FOXP3 kit (Tonbo Biosciences). The staining was performed according to the manufacturer's instructions.
7
Multiparametric Immune Cell Profiling
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Surface and intracellular staining were performed as described previously [27 (link)] using the indicated antibodies. From BioLegend: Alexa fluor 488-Ly6C, PE-Cy7-Ly6G, PE-Cy7-streptavidin, APC-streptatividin; From BD Pharmigen: APC-CD11b, PE-Cy7 anti-mouse CD11c, Rat anti-mouse CD16/CD32, PE-conjugated anti-Gr-1, Alexa 647-conjugated anti- PY418 SRC, PE-conjugated ant-AKT1, from Invitrogen: APC-CD11c, PE-streptavidin, FITC-streptavidin; from AbDSerotec: PE-CD62L; From Santa Cruz Biotechnology: PE-conjugated anti-Fyn. From Cell Signaling, Alexa 647-conjugated anti-SRC rabbit antibody (clone 36D10) and PE-conjugated anti-phospho AKT rabbit antibody (Ser473, clone D9E).
8
Quantification of Regulatory T Cells
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MLN cells or spleen cells were collected (n = 3). After blocking the Fc receptors with purified rat antimouse CD16/CD32 (93; Invitrogen., Tokyo, Japan), frequencies of forkhead box protein 3 (Foxp3) + CD4 + CD25 + Tregs were determined using the Anti-Mouse/Rat Foxp3 Staining Set (77-5775-4-; eBioscience, San Diego, CA), allophycocyanin-conjugated anti-Foxp3 mAb (FJK-16s), PE-conjugated anti-CD25 mAb (PC61.5), and FITC-conjugated anti-CD4 mAb (RM4-5). The stained cells were analyzed using a flow cytometer (FACSCalibur; BD Bioscience). Data were processed using CellQuest software (BD Bioscience). Fluorescence staining was analyzed by fluorescence-activated cell sorting using FlowJo software version 10.0 (FLOWJO, LLC, Ashland, OR).
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