To analyze immune cells in the musculoskeletal tissues at 3 and 7 dpi, ipsilateral feet were perfused extensively, skinned, disjointed from the tibia, and digested in RPM11640 medium supplemented with 10% FBS, 15 mM HEPES, 0.5 mg/ml of collagenase (Sigma, C0130) and 10 ug/ml of DNase I (Sigma, D5025) for 1 h at 37°C. Digested tissue was passed through a 70 μm strainer, and cells were separated by centrifugation and washed with PBS supplemented with 2% FBS and 2 mM EDTA. Cells were stained with a Fixable Viability Dye eFluor 506 (eBioscience, 65-0866-14) and incubated with the following antibodies for 1 h at 4°C: CD16/32 (Biolegend, 101301, 1:200) CD11b PE-Dazzle 594 (BioLegend, 101256, 1:200), Ly6G PerCP-Cy5.5 (Biolegend, 127616, 1:400), Ly6C Pacific Blue (BioLegend, 128014, 1:200), F4/80 APC (Thermo Fisher, 17-4801-82, 1:200), CD11c PE-Cy7 (BD Biosciences, 558079, 1:200), I-A/I-E (MHC class II) Alexa Fluor 700 (BioLegend, 107622, 1:200), NK-1.1 PE (BioLegend, 108708, 1:200), CD3 BUV737 (BD Biosciences, 564380, 1:200), CD8 PerCP-Cy5.5 (BioLegend, 100734, 1:200), CD4 BV786 (BioLegend, 100552, 1:200), B220 BV711 (BioLegend, 103255, 1:200). Cells were analyzed using a BD X20 Fortessa flow cytometer, and all data were processed using FlowJo software (FlowJo, LLC).
Ly6c pacific blue
Manufactured by BioLegend
Sourced in United States
Ly6C Pacific Blue is a fluorescent antibody that binds to the Ly6C antigen. Ly6C is a differentiation antigen expressed on various mouse cell types, including monocytes and granulocytes. The Pacific Blue fluorochrome is used to label the Ly6C antibody, allowing for the detection and analysis of Ly6C-positive cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase, by Merck Group (3 mentions)
Nk1.1 pe, by BioLegend (3 mentions)
Cd45 buv395, by BD (3 mentions)
Cd19 bv605, by BioLegend (3 mentions)
Cd11b percp cy5, by BioLegend (3 mentions)
Dnase 1, by Merck Group (2 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (2 mentions)
F4 80 apc, by Thermo Fisher Scientific (2 mentions)
Cd11c pe cy7, by BD (2 mentions)
Cd11b pe dazzle 594, by BioLegend (2 mentions)
Ly6g percp cy5, by BioLegend (2 mentions)
Cd3 buv737, by BD (2 mentions)
Cd4 bv786, by BioLegend (2 mentions)
B220 bv711, by BioLegend (2 mentions)
Cd8 percp cy5, by BioLegend (2 mentions)
Cd16 32, by BioLegend (2 mentions)
Fortessa x20, by BD (2 mentions)
Ly6g pe cy7, by BioLegend (2 mentions)
Cd3 apc cy7, by BioLegend (2 mentions)
Macsquant analyzer, by Miltenyi Biotec (2 mentions)
6 protocols using ly6c pacific blue
1
Immune Cell Analysis in Musculoskeletal Tissue
2
Immune cell profiling of quadriceps muscle
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Perfused ipsilateral quadriceps muscles were minced and digested in RPMI supplemented with 10% HI-FBS, HEPES pH 7.3, 100 U/ml penicillin and streptomycin, collagenase (2.5 mg/ml; Sigma), and DNase I (30 μg/ml; Roche) in a total of 5 ml at 37°C for 1.5 h. Digested tissue was pressed through a 70 μm cell strainer, and resuspended in RPMI supplemented with 10% HI-FBS and penicillin and streptomycin. Cells were counted using precision count beads (BioLegend). Single cell suspensions were blocked for FcγR binding (BioLegend; clone S17011E) and stained with the following antibodies: CD3 BV421 (BioLegend; clone 145-2C11), CD4 FITC (BioLegend; clone RM4-5), CD8α APC (BioLegend; clone 53–6.3), NK1.1 PE (BioLegend; clone PK136), CD45 BUV395 (BD Biosciences; clone 30-F11), CD19 BV605 (BioLegend; clone 6D5), CD11b PerCP-Cy5.5 (BioLegend; clone M1/70), Ly6C Pacific Blue (BioLegend; clone HK1.4), Ly6G phycoerythrin (PE)-Cy7 (BioLegend; clone 1A8), MHC class II Alexa Fluor 700 (BioLegend; clone M5/114.15.2), Ly6B FITC (BioLegend; clone 7/4), and F4/80 BV650 (BioLegend; clone BM8). Viability was determined by exclusion of a fixable viability dye (eBiosciences; e506). Samples were run on a BD LSRFortessa X-20 flow cytometer and analyzed using FlowJo version 10 (FlowJo, LLC).
3
Isolation and Staining of Myeloid Cells
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For cell depletion experiments, whole blood was harvested from mice and mixed with 5 mM EDTA (Corning). Red blood cells were lysed with ACK lysis buffer at room temperature before being washed and chilled in PBS + 5% FCS. Monocytes were stained with CD45 BUV395 (BD Biosciences clone 30-F11), CD11b PerCP-Cy5.5 (BioLegend clone M1/70), Ly6B FITC (Abcam clone 7/4), Ly6G PE-Cy7 (BioLegend clone 6D5), Ly6C Pacific Blue (BioLegend clone HK1.4) and MHC class II A700 (BioLegend clone M5/114.15.2). NK cells were stained with CD45 BUV95, CD3 APC-Cy-7 (BioLegend clone 145–2C11), CD19 BV605 (BioLegend clone 6D5), and NK1.1 Pe-Cy7 (BioLegend clone PK136). Viability was determined through exclusion of a fixable viability dye (e506;eBiosciences). Samples were fixed and processed on a BD-Fortessa X20. For FcγR expression experiments BV2, LADMAC, and Vero cells were stained with one of the following: CD64 APC (BioLegend clone X54–5/7.1), CD32b APC (Invitrogen clone AT130–2), CD16 FITC (BioLegend clone 221–3A4), CD16.2 APC (BioLegend clone 9E9). Cells were analyzed on a MACSQuant analyzer (Miltenyi). All FACS data were analyzed by FlowJo v. 10.7.
4
Immune Cell Analysis in Musculoskeletal Tissue
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To analyze immune cells in the musculoskeletal tissues at 3 and 7 dpi, ipsilateral feet were perfused extensively, skinned, disjointed from the tibia, and digested in RPM11640 medium supplemented with 10% FBS, 15 mM HEPES, 0.5 mg/ml of collagenase (Sigma, C0130) and 10 ug/ml of DNase I (Sigma, D5025) for 1 h at 37°C. Digested tissue was passed through a 70 μm strainer, and cells were separated by centrifugation and washed with PBS supplemented with 2% FBS and 2 mM EDTA. Cells were stained with a Fixable Viability Dye eFluor 506 (eBioscience, 65-0866-14) and incubated with the following antibodies for 1 h at 4°C: CD16/32 (Biolegend, 101301, 1:200) CD11b PE-Dazzle 594 (BioLegend, 101256, 1:200), Ly6G PerCP-Cy5.5 (Biolegend, 127616, 1:400), Ly6C Pacific Blue (BioLegend, 128014, 1:200), F4/80 APC (Thermo Fisher, 17-4801-82, 1:200), CD11c PE-Cy7 (BD Biosciences, 558079, 1:200), I-A/I-E (MHC class II) Alexa Fluor 700 (BioLegend, 107622, 1:200), NK-1.1 PE (BioLegend, 108708, 1:200), CD3 BUV737 (BD Biosciences, 564380, 1:200), CD8 PerCP-Cy5.5 (BioLegend, 100734, 1:200), CD4 BV786 (BioLegend, 100552, 1:200), B220 BV711 (BioLegend, 103255, 1:200). Cells were analyzed using a BD X20 Fortessa flow cytometer, and all data were processed using FlowJo software (FlowJo, LLC).
5
Flow Cytometry Analysis of Immune Cells
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Blood was collected in tubes containing EDTA. Once the hypothalamus was collected in HBSS, tissue was disaggregated and filtered in a membrane of 40 µm. Next, the labeling of the surface antigens was carried out for 30 min at 4 °C. The surface antibodies and their fluorescences used were: CD206-FITC, CD11-PE, F4/80-PECY7, CD11B-APC, Ly6G-APC-CY7, Ly6C-PacificBlue and CD45-V500 (Biolegend, San Diego, CA, USA). To distinguish between living and dead cells we used 7-aminoactinomycin labeling (Biolegend). After labeling, samples were incubated for 20 min with Quick Lysis buffer (Cytognos, Salamanca, Spain). Samples were analyzed in a BD FACSCanto II flow cytometer (Becton Dickinson, New York, NY, USA) using FACSDiva software. The flow cytometer settings and samples were prepared according to the manufacturer’s instructions. The intensity of surface expression was measured as MFI. The positive stained cells were expressed as percentage values.
6
Isolation and Staining of Myeloid Cells
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