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Ly6c pe hk1.4

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Ly6C-PE (HK1.4) is a fluorescently-labeled antibody used for the identification and characterization of Ly6C-positive cells. It is a component of the Ly6C family of glycosylphosphatidylinositol-anchored proteins. The antibody is conjugated to the fluorescent dye phycoerythrin (PE), allowing for the detection and analysis of Ly6C-expressing cells by flow cytometry.

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Lab products found in correlation

Cd45 bv510 30 f11, by BioLegend (5 mentions) Facscanto 2, by BD (5 mentions) Cd4 bv421 rm4 5, by BioLegend (4 mentions) Cd11b pe cy7, by BioLegend (4 mentions) Cd44 pe im7, by BioLegend (3 mentions) Fc block 2.4g2, by BD (3 mentions) Cd3 apc 17a2, by BioLegend (3 mentions) Ly6g apc 1a8, by BioLegend (3 mentions) Cd8 percpcy5.5 53 6 7, by BioLegend (3 mentions) Gr1 apc cy7 rb6 8c5, by BioLegend (3 mentions) Cd62l fitc mel 14, by BioLegend (2 mentions) B220 apc cy7 ra3 6b2, by BioLegend (2 mentions) Cd8a percp cy5.5 53 6 7, by BioLegend (2 mentions) Cd49d af488 r1 2, by BioLegend (2 mentions) Lsrfortessa, by BD (2 mentions) Cd11c percp cy5, by BioLegend (2 mentions) Ia ie bv421, by BioLegend (2 mentions) Fc block, by BD (2 mentions) F4 80 fitc bm8, by BioLegend (1 mentions) Il 10 pe, by BD (1 mentions)

7 protocols using ly6c pe hk1.4

1

Multiparameter Flow Cytometry for Immune Cell Profiling

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Single-cell suspensions in FACs buffer were incubated in Fc block (2.4G2, BD Biosciences, Franklin Lakes, NJ, USA) for 15 min then samples were washed in FACs buffer and centrifuged at 400 × g for 4 min. Cells were then incubated in a solution containing fluorescently labelled antibodies for 30 min in the dark on ice. Following staining, flow cytometry was performed on a BD FACS Canto II (BD Biosciences) or a LSRFortessa™ (BD Biosciences). Analysis was performed on FlowJo software (BD Biosciences) using the gating strategy outlined in Additional file 2: Fig S2.
The following antibodies were used for the detection and phenotyping of immune cells: CD45-BV510 (30-F11; Biolegend, San Diego, CA, USA), CD3-APC (17A2; Biolegend), B220-APC-Cy7 (RA3-6B2; Biolegend), CD4-BV421 (RM4-5; Biolegend), CD8a-PerCP-Cy5.5 (53–6.7; Biolegend), CD62L-FITC (MEL-14; Biolegend), CD44-PE (IM7; Biolegend), CD11b-PE-Cy7 (M1/70; Biolegend), Ly6G-APC (1A8; Biolegend), Ly6C-PE (HK1.4; Biolegend), and CD49d-AF488 (R1-2; Biolegend).
Peck T., Davis C., Lenihan-Geels G., Griffiths M., Spijkers-Shaw S., Zubkova O.V, & La Flamme A.C. (2023). The novel HS-mimetic, Tet-29, regulates immune cell trafficking across barriers of the CNS during inflammation. Journal of Neuroinflammation, 20, 251.
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2

Multiparametric Flow Cytometry of Immune Cells

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Cells were incubated with Fc Block (1 µg/106 cells; 2.4G2; BD Biosciences) for 15 min. Extracellular staining was performed for 30 min on ice using: CD4‐BV521 (RM4‐5; BioLegend, San Diego, CA, USA), CD45‐BV510 (30‐F11; BioLegend), CD3‐APC (17.A2, BioLegend), CD25‐PE‐Cy7 (PC61; BioLegend), CD8‐PerCPCy5.5 (53‐6.7; BioLegend), B220 (RA3‐6B2; BD Biosciences), CD11b‐PE‐Cy7 (M1/70; BioLegend), Ly6C‐PE (HK1.4; BioLegend), Gr1‐APC‐Cy7 (RB6‐8C5, BioLegend), IA/IE‐BV421 (M5/114.15.2, BioLegend), F4/80‐FITC (BM8, BioLegend), CD11c‐PerCPCy5.5 (N418, BioLegend).
After staining for extracellular proteins, cells were fixed in 4% PFA and permeabilised using 0.1% saponin buffer containing 0.1% bovine serum albumin. Intracellular cytokines were detected with IFNγ‐BV421 (XMG 1.2, BioLegend), IL‐10‐PE (54902, BD Bioscience), and IL‐17A‐AF647 (TC11‐18H10, BioLegend).
Flow cytometry was performed on a BD FACS Canto II (BD Biosciences) and analysed using FlowJo software version 10.6 (Treestar Inc., Ashland, OR, USA).
Denny L., Al Abadey A., Robichon K., Templeton N., Prisinzano T.E., Kivell B.M, & La Flamme A.C. (2021). Nalfurafine reduces neuroinflammation and drives remyelination in models of CNS demyelinating disease. Clinical & Translational Immunology, 10(1), e1234.
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3

Flow Cytometry Analysis of Bone Marrow and Spleen Cells

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Bone marrow and spleen cells were stained for flow cytometry as previously described (Mayer et al., 2017 (link)). Gating strategy for BM erythroid cells was described in the Supplementary Figure 1. Cells were stained with the following antibodies: CD45-FITC (30-F11, eBioscience), CD11b-FITC (M1/70, eBioscience), Gr-1-FITC (RB6-8C5, eBioscience), Ter119-APC (TER119, Biolegend) and CD44-PE (IM7, Biolegend), F4/80-APC (BM8, Biolegend), Gr-1-FITC (RB6-8C5, Biolegend), CD3-PE (145-2C11, Biolegend), Ly6C-PE (HK1.4, Biolegend), CD106-PE (429, Biolegend), CD45R/B220-APC (RA3.682, Biolegend). Cells were analyzed using AccuriC6 flow cytometer and data were analyzed using FlowJo software (v10.7.1).
Myneni V.D., Szalayova I, & Mezey E. (2021). Differences in Steady-State Erythropoiesis in Different Mouse Bones and Postnatal Spleen. Frontiers in Cell and Developmental Biology, 9, 646646.
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4

Multiparametric Analysis of Immune Cells

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Cells were incubated with Fc Block (1 μg/106 cells; 2.4G2; BD Biosciences) for 15 min. Extracellular staining was performed for 30 min on ice using the following antibodies: CD4-BV421 (RM4-5; BioLegend, San Diego, CA, USA), CD45-BV510 (30-F11; BioLegend), CD3-APC (17.A2, BioLegend), CD25-PE-Cy7 (PC61; BioLegend), CD8 PerCPCy5.5 (53-6.7; BioLegend), B220-APC-Cy7 (RA3-6B2; BD Biosciences), CD11b-PE-Cy7 (M1/70; BioLegend), Ly6C-PE (HK1.4; BioLegend), Gr1-APC-Cy7 (RB6-8C5; BioLegend), IA/IE-BV421 (M5/114.15.2; BioLegend), F4/80-FITC (BM8; BioLegend), CD11c-PerCP-Cy5.5 (N418; BioLegend).
After staining for extracellular proteins, cells were fixed in 4% paraformaldehyde (PFA, pH 7.4) and permeabilized using 0.1% saponin buffer containing 0.1% bovine serum albumin. For intracellular cytokine detection, interferon-γ (IFNγ)-BV421 (XMG 1.2, BioLegend), interleukin (IL)-10-PE (54902, BD Bioscience), and IL-17A-AF647 (TC11-18H10, BioLegend) antibodies were used.
Flow cytometry was performed on a BD FACS Canto II (BD Biosciences) and analyzed using FlowJo software version 10.1 (Treestar Inc., Ashland, OR, USA).
Paton K.F., Robichon K., Templeton N., Denny L., Al Abadey A., Luo D., Prisinzano T.E., La Flamme A.C, & Kivell B.M. (2021). The Salvinorin Analogue, Ethoxymethyl Ether Salvinorin B, Promotes Remyelination in Preclinical Models of Multiple Sclerosis. Frontiers in Neurology, 12, 782190.
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5

Multiparameter Flow Cytometry Immunophenotyping

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For the detection of immune cells, the following antibodies were used: CD4-BV421 (RM4-5; Biolegend, San Diego, CA, USA), CD45-BV510 (30-F11; Biolegend), CD25-AF488 (PC61; Biolegend), CD8-PerCPCy5.5 (53-6.7; Biolegend), CD11b-PE-Cy7 (M1/70; Biolegend), CD3-APC-Cy7 (17A2; Biolegend), Ly6C-PE (HK1.4; Biolegend), Ly6G-APC (1A8; Biolegend), CD45R-AF488 (RA3-6B2; BD Bioscience), CCR2-PE (475,301; R&D), CCR5-APC (HM-CCR5; Biolegend), and Gr1-APC-Cy7 (RB6-8C5, Biolegend). Cells were incubated with Fc Block (2.4G2; BD Biosciences) for 15 min prior to staining with fluorescently labeled antibodies for 30 min on ice. Flow cytometry was performed on a BD FACS Canto II (BD Biosciences) and analyzed using Flowjo software version 10.1 (Treestar Inc., Ashland, OR, USA).
Robichon K., Patel V., Connor B, & La Flamme A.C. (2020). Clozapine reduces infiltration into the CNS by targeting migration in experimental autoimmune encephalomyelitis. Journal of Neuroinflammation, 17, 53.
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6

Flow Cytometric Immunophenotyping of Leukocytes

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Before staining, cells were incubated with FcBlock reagent (anti-CD16/CD32, Biolegend, Ozyme-France) to block the FcγRII/III receptors. The cellular content of the peritoneal exudates was characterized using a combination of fluorochrome conjugated mAbs: CD45-vioblue (REA737, Miltenyi Biotec), Ly6G-FITC (1A8, Miltenyi Biotec) F4/80-APC (CI:A3-1; AbD Serotec, BioRad, Luxembourg), Ly6C-PE (HK1.4, Biolegend), and CCR3-APC-Fire (J073E5, Biolegend). At analysis, doublets and dead cells, labeled with 7-AAD (Biolegend), were excluded. All samples were acquired using a MACS Analyzer (Miltenyi Biotec) flow cytometer and analyzed using FlowJo software (TreeStar, USA). The gating strategies are presented in Supplementary Material. Mammary gland cells were labeled with the following fluorochrome conjugated mAbs: anti-CD45-PECy7 (I3/2.3; Southern Biotech), Anti-Ly6G-FITC (1A8, Miltenyi Biotec), and anti-F4/80-APC (CI:A3-1; AbD Serotec) to identify leukocytes, neutrophils, and macrophages, respectively. At analysis, doublets, red blood cells, and lymphocytes were excluded based on CD45 staining. All samples were acquired using a CytoFLEX (Beckman Coulter, USA) flow cytometer and analyzed using CytExpert software (Beckman Coulter, USA).
Walachowski S., Breyne K., Secher T., Cougoule C., Guzylack-Piriou L., Meyer E., Foucras G, & Tabouret G. (2022). Oral supplementation with yeast β-glucans improves the resolution of Escherichia coli-associated inflammatory responses independently of monocyte/macrophage immune training. Frontiers in Immunology, 13, 1086413.
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7

Multiparameter Flow Cytometry for Immune Cell Profiling

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Single-cell suspensions in FACs buffer were incubated in Fc block (2.4G2, BD Biosciences, Franklin Lakes, NJ, USA) for 15 min then samples were washed in FACs buffer and centrifuged at 400 × g for 4 min. Cells were then incubated in a solution containing fluorescently labelled antibodies for 30 min in the dark on ice. Following staining, flow cytometry was performed on a BD FACS Canto II (BD Biosciences) or a LSRFortessa™ (BD Biosciences). Analysis was performed on FlowJo software (BD Biosciences) using the gating strategy outlined in Additional file 2: Fig S2.
The following antibodies were used for the detection and phenotyping of immune cells: CD45-BV510 (30-F11; Biolegend, San Diego, CA, USA), CD3-APC (17A2; Biolegend), B220-APC-Cy7 (RA3-6B2; Biolegend), CD4-BV421 (RM4-5; Biolegend), CD8a-PerCP-Cy5.5 (53–6.7; Biolegend), CD62L-FITC (MEL-14; Biolegend), CD44-PE (IM7; Biolegend), CD11b-PE-Cy7 (M1/70; Biolegend), Ly6G-APC (1A8; Biolegend), Ly6C-PE (HK1.4; Biolegend), and CD49d-AF488 (R1-2; Biolegend).
Peck T., Davis C., Lenihan-Geels G., Griffiths M., Spijkers-Shaw S., Zubkova O.V, & La Flamme A.C. (2023). The novel HS-mimetic, Tet-29, regulates immune cell trafficking across barriers of the CNS during inflammation. Journal of Neuroinflammation, 20, 251.
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