The flow cytometry protocol for immune cells used as previously described17 . Samples for flow cytometry analysis were dissected and processed following Miltenyi Biotec protocol for neural tissue dissociation kit (Miltenyi Biotec, Auburn, CA, USA). Briefly, spinal cords from spina bifida fetuses exposed and nonexposed and sham animals were harvested and digested with papain enzyme at 37 °C in rotation using gentle MACS Dissociator (Miltenyi Biotec). Cell suspension was meshed with 70-µm Strainer (BD Pharmingen, San Jose, CA) and labeled at 4 °C for 30 min with a specific antibodies panel previously reported17 for flow cytometry. Cell populations were analyzed by flow cytometry on a LSRII Flow Cytometer system (BD Pharmingen) depending the immunolabeling for microglia/macrophages (CD45/CD11b/c+), and activated microglia (CD200R+ and MHCII+). Antibodies used for flow cytometry experiment were: Fixable viability staining-APC-eFluor780 (eBioscience, Rockford, IL), CD45-PECy7 (Biolegend, San Diego, CA), CD11b/c-PerCP-Cy5.5 (Biolegend), CD200R-FITC (Biolegend), MHCII-PE (eBioscience). Data were analyzed using FlowJo software (v.10).
Cd200r fitc
Manufactured by BioLegend
Sourced in United States
CD200R-FITC is a fluorescently-labeled monoclonal antibody that binds to the CD200 receptor on the surface of cells. It is designed for use in flow cytometry applications to identify and quantify cells expressing the CD200 receptor.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto, by BD (2 mentions)
Cd86 apc, by BioLegend (2 mentions)
Apc efluor 780, by Thermo Fisher Scientific (1 mentions)
Lsr 2 flow cytometer system, by BD (1 mentions)
Cd45 pe cy7, by BioLegend (1 mentions)
Mhcii pe, by Thermo Fisher Scientific (1 mentions)
70 m strainer, by BD (1 mentions)
Macs dissociator, by Miltenyi Biotec (1 mentions)
Neural tissue dissociation kit, by Miltenyi Biotec (1 mentions)
Cd62l apc, by BioLegend (1 mentions)
Fitc il 4, by Thermo Fisher Scientific (1 mentions)
Fcεriα fitc, by BioLegend (1 mentions)
Ccr7 apc, by BioLegend (1 mentions)
Cd63 apc, by BioLegend (1 mentions)
Cd203c pe, by BioLegend (1 mentions)
Il 4 pecy7, by Thermo Fisher Scientific (1 mentions)
Il 6 apc, by Thermo Fisher Scientific (1 mentions)
Lysys 2 software, by BD (1 mentions)
Il 6 fitc, by Thermo Fisher Scientific (1 mentions)
4 protocols using cd200r fitc
1
Characterizing Immune Cells in Spina Bifida
2
Basophil Activation Profiling in Humans and Mice
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Human basophils were gated on FcεRIα-FITC/CD123-PerCP/Cy5.5/CD203c-PE (BioLegend, San Diego, CA, USA) positive cells after extracellular and intracellular staining. The expression levels of CD203c-PE, CD62L-APC, FcεRIα-FITC, CCR7-APC, CD63-APC, IL-13-APC, B cell-activating factor (BAFF)-APC (BioLegend, San Diego, CA, USA), IL-4-PE-Cy7, and IL-6-APC (eBioscience, San Diego, CA, USA) in basophils were quantified and expressed as relative fluorescence units (the ratio of mean fluorescence intensity normalized to controls) or as a positive percentage of total basophils.
Mouse basophils were gated on CD49b-APC/IgE-PE (BioLegend, San Diego, CA, USA). The expression levels of the activation marker CD200R-FITC (BioLegend, San Diego, CA, USA) (26 (link)), IL-4-FITC, and IL-6-FITC (eBioscience, San Diego, CA, USA) were quantified and expressed in the same way as for human basophils. A FACScanto™ Π flow cytometer (Becton Dickinson, San Jose, CA, USA) and Lysys II software (Becton Dickinson, San Jose, CA, USA) or FlowJo Software (Tree Star, San Carlos, CA, USA) were used to acquire and analyze the data.
Mouse basophils were gated on CD49b-APC/IgE-PE (BioLegend, San Diego, CA, USA). The expression levels of the activation marker CD200R-FITC (BioLegend, San Diego, CA, USA) (26 (link)), IL-4-FITC, and IL-6-FITC (eBioscience, San Diego, CA, USA) were quantified and expressed in the same way as for human basophils. A FACScanto™ Π flow cytometer (Becton Dickinson, San Jose, CA, USA) and Lysys II software (Becton Dickinson, San Jose, CA, USA) or FlowJo Software (Tree Star, San Carlos, CA, USA) were used to acquire and analyze the data.
3
Microglia Polarization Analysis
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For analysis of microglia polarization, the microglia were incubated at room temperature for 30 min with the uorescently labeled antibodies CD86-APC (1:200, BioLegend, San Diego, USA) and CD200R-FITC (1:200, BioLegend). After incubation, the cells were washed, centrifuged and then resuspended in PBS to a nal volume of 500 μL. Flow cytometry was performed on a FACSCanto apparatus (BD Biosciences, Bedford, USA). The obtained data were analyzed using FlowJo software (version 10.1, BD Biosciences).
4
Microglia Polarization Analysis
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For analysis of microglia polarization, the microglia were incubated at room temperature for 30 min with the uorescently labeled antibodies CD86-APC (1:200, BioLegend, San Diego, USA) and CD200R-FITC
(1:200, BioLegend). After incubation, the cells were washed, centrifuged and then resuspended in PBS to a nal volume of 500 μL. Flow cytometry was performed on a FACSCanto apparatus (BD Biosciences, Bedford, USA). The obtained data were analyzed using FlowJo software (version 10.1, BD Biosciences).
(1:200, BioLegend). After incubation, the cells were washed, centrifuged and then resuspended in PBS to a nal volume of 500 μL. Flow cytometry was performed on a FACSCanto apparatus (BD Biosciences, Bedford, USA). The obtained data were analyzed using FlowJo software (version 10.1, BD Biosciences).
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