Single cell suspensions of splenocytes were generated from 6-7-week-old pre-diabetic NOD.Foxp3-GFP/cre mice (Stock No: 008694, Jackson Laboratory), followed by RBC lysis. Cells were resuspended at 106/mL in cRPMI and treated with 10 IU/mL rh-IL-2 (Teceleukin) and/or 100 ng/mL rhIGF1 (BioVision) for two days at 37°C. Live/Dead fixable near-IR dead cell stain kit (Invitrogen) was applied according to manufacturer’s instructions for dead cell exclusion. Cells were washed once with stain buffer and then, incubated with anti-CD16/32 (Clone 2.4G2, BD Biosciences) for 5 minutes at 4°C. Samples were stained with the following fluorescently-labeled anti-mouse antibodies at 4°C for 30 minutes: CD4-PerCP-Cy5.5 (RM4-5, eBioscience), CD62L-BV510 (MEL-14, BioLegend), CD44-PE-Cy7 (IM7, BioLegend), CD221-PE (3B7, Santa Cruz Biotechnology), CD25-AF700 (PC61), CD122-PE-Dazzle594 (TM-β1), CD132-APC (TUGm2) (BioLegend). Samples were fixed and permeabilized with Foxp3 transcription factor staining buffer set according to manufacturer’s protocol (eBioscience) before staining for 45 minutes at 23°C with anti-mouse Foxp3-AF488 (FJK-16s, eBioscience) and Helios-Pacific Blue (22F6, BioLegend). Samples were washed once with stain buffer prior to data acquisition on a Cytek Aurora 5L (16UV-16V-14B-10YG-8R) spectral flow cytometer and analysis with FlowJo software (v10.6.1; Tree Star).
Cd4 percp cy5.5 rm4 5
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD4 PerCp-Cy5.5 (RM4-5) is a fluorescent-conjugated antibody used for the detection and analysis of CD4+ T cells in flow cytometry applications. It is a PerCp-Cy5.5 conjugated anti-mouse CD4 monoclonal antibody.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b pe cy7 m1 70, by Thermo Fisher Scientific (2 mentions)
Facsvia flow cytometry system, by BD (2 mentions)
Gr1 pe rb6 8c5, by BioLegend (2 mentions)
Foxp3 transcription factor staining buffer, by Thermo Fisher Scientific (1 mentions)
Cd44 pe cy7 im7, by BioLegend (1 mentions)
Cd221 pe 3b7, by Santa Cruz Biotechnology (1 mentions)
Cd62l bv510 mel 14, by BioLegend (1 mentions)
Helios pacific blue 22f6, by BioLegend (1 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Anti cd16 32 clone 2.4g2, by BD (1 mentions)
Cytofix cytoperm plus fixation permeabilization kit, by BD (1 mentions)
Interferon γ apc xmg1.2, by Thermo Fisher Scientific (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Foxp3 apc fjk 16s, by Thermo Fisher Scientific (1 mentions)
Fc block 2.4g2, by BD (1 mentions)
Cd8a pe cy7 53 6.7, by BioLegend (1 mentions)
Nk1.1 apc pk136, by Thermo Fisher Scientific (1 mentions)
Ly6c pe hk1.4, by Thermo Fisher Scientific (1 mentions)
Ly6g bv421, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
5 protocols using cd4 percp cy5.5 rm4 5
1
Characterizing Pre-Diabetic NOD Splenocytes
2
Multi-parametric Flow Cytometry for T-cell and Regulatory Immune Responses
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ICS for T-cell cytokines was performed as described,2 (link) using the Cytofix/Cytoperm Plus fixation/permeabilization kit (BD Biosciences), and antibodies to CD4 PE-Cy7 (RM4-5), IL-17A PerCP-Cy5.5 (eBio17B7), and interferon-γ APC (XMG1.2) (all eBioscience). For staining of regulatory T cells (Treg), the FoxP3 staining buffer set (eBioscience) and antibodies to CD4 PerCP-Cy5.5 (RM4-5), CD25 APC (PC61.5), and FoxP3 PE (FJK-16 s) were used. Intracellular IL-10 expression was analyzed by flow cytometry as previously described,9 (link) using the Cytofix/Cytoperm Plus fixation/permeabilization kit (BD Biosciences). The following antibodies were used: CD19 PerCP-Cy5.5 (eBio1D3), CD45 PE-Cy7 (30-F11), CD11b APC-Cy7 (M1/70), IL-10 APC (JES5-16E3) (all from eBioscience), and B220 (CD45R) FITC (RA3-6B2) (BD Biosciences). An isotype- and fluorochrome-matched control antibody (IgG2b κ APC; eBioscience) was used to assess nonspecific staining for IL-10.
3
Comprehensive Flow Cytometry Analysis
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Flow cytometry analyses were carried out using a two-laser standard configuration BD FACSVia™ flow cytometry system. We analyzed 6–10 mice for each experimental group. Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The gates were set using fluorescence minus one (FMO,) control strategy. FMO controls are samples that include all conjugated Abs present in the test samples except one. The channel in which the conjugated Ab is missing is the one for which the FMO provides a gating control. The following mAbs were used: CD4 PerCp-Cy5.5 (RM4-5, eBioscience, San Diego, CA, USA); CD11b Pe-Cy7 (M1/70, eBioscience); Gr1 PE (RB6-8C5, BioLegend); IL-10 FITC (JES5-16E3, eBioscience) and FoxP3 APC (FJK-16s, eBioscience).
4
Multiparametric Spleen Cell Profiling
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Spleens were processed with frosted glass slides and filtered (70 µm) to create single-cell suspensions. Red blood cells were lysed with ammonium-chloride-potassium lysis buffer for 5 min on ice, and remaining cells were washed with PBS before staining. 106 cells per sample were stained with Fixable Live/Dead Near Infrared (#L34975; Thermo Fisher) for dead cell exclusion. Cells were incubated with Fc block (2.4G2; BD Biosciences) for 5 min on ice before staining with the following antibodies at appropriate concentrations for 30 min on ice: CD4-PerCP-Cy5.5 (RM4-5; eBioscience), CD8a-PE-Cy7 (53-6.7; BioLegend), CD3e-BV605 (145-2C11; BioLegend), NK1.1-APC (PK136; eBioscience), CD19-BV711 (6D5; BioLegend), Ly6G-BV421 (1A8; BD Biosciences), Ly6C-PE (HK1.4; eBioscience), and CD11b-AF488 (M1/70; eBioscience). Samples were washed once before data acquisition on an LSR Fortessa (BD Biosciences) and analysis using FlowJo (v10.5.0; TreeStar).
5
Isolation and Analysis of Colonic Lamina Propria Cells
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The cells were isolated from the colon lamina propria using the Lamina Propria Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany; 130-097-401), according to the instructions.
Flow cytometry analyses were carried out using a two-laser standard configuration BD FACSVia™ flow cytometry system. Data were analyzed using FlowJo software (TreeStar). The gates were set using the fluorescence minus one (FMO) control strategy. FMO controls are samples that include all conjugated Abs present in the test samples except one. The channel in which the conjugated Ab is missing is the one for which the FMO provides a gating control. The following mAbs were used: CD4 PerCp-Cy5.5 (RM4-5, eBioscience, Affymetrix Santa Clara, CA, USA); CD11b Pe-Cy7 (M1/70, eBioscience, Affymetrix, Santa Clara, CA, USA); Gr1 PE (RB6-8C5, BioLegend, San Diego, CA, USA); IL-10 FITC (JES5-16E3, eBioscience, Affymetrix Santa Clara, CA, USA); and FoxP3 APC (FJK-16s, eBioscience, Affymetrix, Santa Clara, CA, USA).
Flow cytometry analyses were carried out using a two-laser standard configuration BD FACSVia™ flow cytometry system. Data were analyzed using FlowJo software (TreeStar). The gates were set using the fluorescence minus one (FMO) control strategy. FMO controls are samples that include all conjugated Abs present in the test samples except one. The channel in which the conjugated Ab is missing is the one for which the FMO provides a gating control. The following mAbs were used: CD4 PerCp-Cy5.5 (RM4-5, eBioscience, Affymetrix Santa Clara, CA, USA); CD11b Pe-Cy7 (M1/70, eBioscience, Affymetrix, Santa Clara, CA, USA); Gr1 PE (RB6-8C5, BioLegend, San Diego, CA, USA); IL-10 FITC (JES5-16E3, eBioscience, Affymetrix Santa Clara, CA, USA); and FoxP3 APC (FJK-16s, eBioscience, Affymetrix, Santa Clara, CA, USA).
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