Mice were anaesthetized with ketamine and xylazine and perfused with ice-cold PBS. Brains were removed and homogenized with a Dounce homogenizer in cold HBSS buffer (Sigma-Aldrich). Brain homogenate was resuspended to prepare 30% isotonic Percoll (Sigma-Aldrich), which was over-layered on 70% isotonic Percoll. The gradient was then centrifuged at 500 g for 25 min at 18°C. Mononuclear cells were collected from the 30/70% interface and washed with PBS. To identify microglia, monocytes and granulocytes, isolated mononuclear cells were first pre-incubated with mouse anti-CD16/32 antibody (1:50; TruStain FcX™, BioLegend) for 15 min at 4°C to block Fc receptors, and then stained with fluorochrome-conjugated antibodies (1:100; APC-CD11b, FITC-CD45, Miltenyi Biotec), FITC-Ly6C (1:50; Invitrogen, Vilnius, Lithuania), V450-Ly6G (1:50; BioLegend). Cells were then rinsed with FACS buffer, run on BD FACS Verse) and analysed using BD FACS Suitev1.0.6 and FlowJoV10 software.
Ly6c fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ly6C-FITC is a fluorochrome-conjugated antibody that binds to the Ly6C surface antigen. It is primarily used for the identification and analysis of Ly6C-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
F4 80 apc, by Thermo Fisher Scientific (2 mentions)
Cd11b pb, by Thermo Fisher Scientific (2 mentions)
Hbss buffer, by Merck Group (1 mentions)
Isotonic percoll, by Merck Group (1 mentions)
Trustain fcx, by BioLegend (1 mentions)
Luminol, by Biosynth (1 mentions)
Cd11c pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd45 percp cy5, by BD (1 mentions)
Anti il 4 fitc, by BD (1 mentions)
1a8 apccy7, by Thermo Fisher Scientific (1 mentions)
Gr1 apc, by Thermo Fisher Scientific (1 mentions)
Cd45 apc, by BD (1 mentions)
Anti ifn γ pe cy7, by BD (1 mentions)
Cd8 apc, by BD (1 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (1 mentions)
Ly6g pacific blue, by BioLegend (1 mentions)
Cd11c alexafluor700, by BioLegend (1 mentions)
Cd4 pe, by Thermo Fisher Scientific (1 mentions)
Cd11b pe cy7, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd16 cd32 fc block, by Thermo Fisher Scientific (1 mentions)
8 protocols using ly6c fitc
1
Isolation and Flow Cytometry of Murine CNS Immune Cells
2
Isolation and Identification of Monocytes and MoMFs
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BMDMs and hepatic non-parenchymal cells were isolated from WT and Fpr2−/− mice (n = 3 per genotype) as previously described [22 (link)]. Cells were then immunostained with PE-F4/80, FITC-Ly6C, and PerCy5.5-Cd11b (Invitrogen, Waltham, MA, USA) to identify the monocyte (F4/80lo, Ly6Chi, Cd11b+) and MoMF populations (F4/80hi, Ly6Clo, Cd11b+) by flow cytometry.
3
Flow Cytometry Analysis of Immune Cells
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Stained cells were analyzed using a BD LSR II-SORP system (Becton Dickinson) and analyzed with FlowJo software (Tree Star). The following mAbs were used: CD45-PerCPCy5, CD45-APC, CD8-APC, anti-IFNγ-PECy7 and anti-IL-4-FITC (BD Biosciences), Gr1-APC, CD11c-PECy7, CD11b-PB, F4/80-APC (eBiosciences), 1A8-APCCy7, Ly6C-FITC, 1A8-APCCy7, CD4-AF700. DAPI and AnV-FITC (BioLegends). In vivo bioluminescence imaging of MPO activity was quantified through injection of 200mg/kg luminol (Carbosynth) i.p. 10 minutes before luminescence acquisition. Photon emission was acquired for 10 minutes using a Xenogen IVIS Imaging system.
4
Comprehensive Immune Cell Profiling
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All cells were stained with Live/Dead Fixable Aqua (1:2000; Invitrogen) at room temperature for 30 min in the dark. Following the Live/Dead viability stain, cells were incubated with anti-mouse CD16/CD32 (Fc block; 1:100, eBioscience), CD3 PE-eFluor610 (1:100; eBioscience), CD4 PE (1:100; eBioscience), CD8b APC-eFluor780 (1:100; eBioscience), CD11b PE-Cy7 (1:200; eBioscience), Ly6G Pacific Blue (1:100; BioLegend), CD45 PerCP-Cy5.5 (1:100; eBioscience), CD11c Alexa Fluor 700 (1:50; BioLegend) Ly6C FITC (1:200; eBioscience) in FACS buffer. Samples were run on an LSRII (BD Biosciences) flow cytometer and analyzed with FlowJo_V10 software.
Single cell lymphocytes were gated on forward scatter area (FSA; size) and side scatter area (SSA; granularity) and then by forward scatter height (FSH) and FSA. Live cells were selected as the negative Fixable Aqua population. CD45+ cells were selected and gated by CD3 (T cells) versus CD11b (myeloid cells). The CD3+ population was then gated on CD4 (helper T cells) and CD8 (cytotoxic T cells). CD11b+ cells were further gated on CD11c and Ly6G for the following populations: CD11c−Ly6G− (microglia and macrophages), CD11c-Ly6G+ (neutrophils), and CD11c+Ly6G− (dendritic cells). CD11c−Ly6G− cells were gated for CD45 and low and high populations were further gated for MHCII and Ly6C to determine their activation state (Suppl. Fig.S1 ).
Single cell lymphocytes were gated on forward scatter area (FSA; size) and side scatter area (SSA; granularity) and then by forward scatter height (FSH) and FSA. Live cells were selected as the negative Fixable Aqua population. CD45+ cells were selected and gated by CD3 (T cells) versus CD11b (myeloid cells). The CD3+ population was then gated on CD4 (helper T cells) and CD8 (cytotoxic T cells). CD11b+ cells were further gated on CD11c and Ly6G for the following populations: CD11c−Ly6G− (microglia and macrophages), CD11c-Ly6G+ (neutrophils), and CD11c+Ly6G− (dendritic cells). CD11c−Ly6G− cells were gated for CD45 and low and high populations were further gated for MHCII and Ly6C to determine their activation state (Suppl. Fig.
5
Multiparameter Flow Cytometry Analysis
6
Isolation and Analysis of MDSCs
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To analyze MDSCs, single-cell suspensions were prepared from peripheral blood, spleens, and hearts. Collected cells were labeled with CD11b (PerCp-cy5.5), Ly6C (FITC), and Ly6G (all from eBioscience, San Diego, CA, USA) and analyzed on BD FACSAria™ II (BD Biosciences). Cell sorting was performed on BD FACSAria™ III Cell Sorter (BD Biosciences).
7
Flow Cytometric Isolation of Joint Cells
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Harvested joints were digested in collagenase/dispase with DNase and shredded with rat-tooth forceps to isolate cells before staining them for flow cytometry as described (22 (link)). About 5x105 cells were incubated in a 96-well U-bottom plate with Fc block (anti-CD16/CD32; eBioscience, San Diego, CA) then surface stained as indicated with the following antibodies: CD45.2 PE, F4/80 APC, Ly-6G PE-Cy7, and Ly-6C FITC (all from eBioscience). Cells were then washed and fixed in 4% paraformaldehyde for 15 minutes. For each sample 50,000 events were analyzed using a BD LSRFortessa X-20 flow cytometer and data analysis was performed using FlowJo 10.8.1 software.
8
Quantification of Immune Cell Populations
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NPC cells (0.3-0.5 × 10 6 cells/test) were incubated for 20 min at RT in the dark with the following antibodies (5 μg/ml): CD11b-Mac1-PECy7 (rat IgG2bk, anti-mouse eBioscience, San Diego CA, USA), CD45-FITC (rat IgG, Beckman), F4/80-PE (ratIgG2ak, eBioscience), Ly6C-FITC (rat IgMk, anti-mouse, Pharmingen, San José, CA, USA) and CCR2-APC (ratIgG2B, R&DSystems, Minneapolis, MN, USA) or their corresponding isotype controls. After three washes, Perfect-Count microspheres (Cytognos, Salamanca, Spain) were added to quantify the exact number of cells. Flow cytometry analysis was performed using Cytomics FC500 with the CXP program. In Supplementary Fig. 1 is shown the efficiency of isolation of immune cell population in RCD and MCD groups.
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