Tissue samples were obtained by cutting the quadriceps femoris muscle from the hip to just above the knee. Tissues were finely diced manually with standard razor blades and digested for 45 min at 37°C with 1.67 Wünsch U/mL (0.5 mg/mL) Liberase TL (Roche Diagnostics, Sigma Aldrich) and 0.2 mg/mL DNase I (Roche Diagnostics, Sigma Aldrich) in RPMI-1640 medium supplemented with L-Glutamine and 15 mM HEPES (Gibco). The digested tissues were then pestled through 70 μm cell strainers (ThermoFisher Scientific) with RPMI-1640 (supplemented as before), and then washed twice with 1X DPBS. A Percoll (GE Healthcare) density gradient centrifugation was used to enrich the leukocyte fraction and remove blood and debris from the muscle samples and centrifuged at 2100 x g for 30 min with the lowest acceleration and no brake. Cells were washed 1X DPBS, stained with a viability live/dead amine reactive dye, washed with 1X DPS, blocked with anti-mouse TruStain FcX (BioLegend), and surface stained (Supplementary Table 6 ). Flow cytometry was performed using an Attune NxT Flow Cytometer (ThermoFisher Scientific) with a Violet, Blue, Green, and Red laser configuration. All subsequent analyses were performed on Live singlet cells using Windows based FlowJo™ software v10 (Benton Dickinson), license supplemented courtesy of the Johns Hopkins Bloomberg Flow Cytometry and Immunology Core.
Anti mouse trustain fcx
Manufactured by BioLegend
Sourced in United States
Anti-mouse TruStain FcX is a Fc receptor blocking reagent designed to reduce non-specific antibody binding in mouse samples. It functions by blocking Fc receptors to minimize background staining in flow cytometry and other immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
70 μm cell strainer, by Thermo Fisher Scientific (3 mentions)
Liberase tl, by Roche (3 mentions)
Percoll, by GE Healthcare (3 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Roche (3 mentions)
Facsaria 2, by BD (2 mentions)
Sca 1 pe cy7, by Thermo Fisher Scientific (2 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Cd3 af488, by BioLegend (2 mentions)
True nuclear transcription factor buffer set, by BioLegend (2 mentions)
Cell stain buffer, by BioLegend (2 mentions)
Cd45 1 percp cy5, by BioLegend (2 mentions)
Cd45.2 pacific blue, by BioLegend (2 mentions)
Lsrfortessa cell analyser, by BD (1 mentions)
Sytox blue dead cell stain, by Thermo Fisher Scientific (1 mentions)
Cd23 clone b3b4, by BD (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Ccr2 pe, by R&D Systems (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti mouse trustain fcx
1
Muscle Tissue Leukocyte Enrichment
2
Multicolor Flow Cytometry Phenotyping
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Antibodies against the following antigens were used: anti-mouse B220 (clone 6B2), CD38 (clone 90), IgM (clone 11–41), CD24 (clone M1/69), CD21/35 (clone 7E9), CD23 (clone B3B4), and IgD (clone 11-26c) from BD Biosciences. Anti-mouse CD5 (clone 53-7.3) and CD11b (clone M1/70) from Biolegend. Anti-mouse CD11a (integrin αL, clone M17/4), CD18 (integrin β2, clone M18/2), CD29 (integrin β1, clone HMb1-1), and CD49d (integrin α4, clone R1-2) from eBioscience. Murine cells were resuspended in DPBS + 3% FCS and stained for 30 min at 4°C with different anti-mouse antibodies in the presence of anti-mouse TruStain fcX (Biolegend). Control samples were labeled with matched isotype control antibodies. The samples were resuspended in Sytox Blue Dead Cell Stain (1:1,000 in DPBS; Invitrogen) before acquisition. Samples were acquired by flow cytometry (BD LSRFortessa cell analyser, Becton Dickinson) with FACSDiva software v6.2 (Becton and Dickinson) and data were analyzed using FlowJo (Tree Star) software.
3
Monocyte Phenotyping by Flow Cytometry
4
Isolation and Purification of Mouse Bone Marrow Cell Subsets
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Single cell suspensions of fresh BMCs extracted from non-treated mice were used to purify various BMC subpopulations. For hematopoietic stem cells (HSCs) and myeloid cell precursors, lineage-specific cells were first eliminated by magnetic cell sorting using the Mouse Hematopoietic Progenitor Cell Isolation kit (Stemcell Technologies, Vancouver, BC, Canada) as per the supplied protocol. Depleted cells were used at a concentration of under 1x106 cells/100 μL. Fc receptors were blocked by adding 1 μg/100 μL of anti-mouse TruStain FcX (Biolegend) for 10 minutes at room temperature. Cells were then stained with anti-Sca-1 and anti-CD34 (Biolegend). HSCs were identified as Lin-Sca-1+CD34- cells, while precursors were identified as Lin-Sca-1-CD34+ cells. For lineage-specific immune cell types, separate BMC extractions were used to sort monocytes (CD11b+Ly6G-), neutrophils (CD11b+Ly6G+) and B-lymphocytes (CD19+). Low FSC events were always discriminated against while sorting to eliminate cell debris and microparticles. All samples were processed using a MoFlo XDP. A small portion of sorted cells were analyzed using the FoFlo XDP to ensure the efficiency of the sorting.
5
Multiparameter Flow Cytometry Analysis
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Single cell suspensions from both mice and human samples were first stained with Zombie Violet (BioLegend, Cat#423114) and then washed 2x with PBS prior to incubation with anti-mouse TruStain FcX™ (BioLegend, Cat#101319) or anti-human TruStain FcX™ (BioLegend, Cat# 422301), respectively, to block nonspecific Fc domain binding. Cells were then stained with the desired antibodies listed in Supplementary Tables 1 2 Supplementary Tables 1 2
6
Muscle Tissue Leukocyte Enrichment
7
Isolation and Characterization of Muscle-Derived Leukocytes
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Tissue samples were obtained by cutting the quadriceps femoris muscle from the hip to just above the knee. Tissues were finely diced manually with standard razor blades and digested for 45 min at 37 o C with 1.67 Wünsch U/mL (0.5 mg/mL) Liberase TL (Roche Diagnostics, Sigma Aldrich) and 0.2 mg/mL DNase I (Roche Diagnostics, Sigma Aldrich) in RPMI-1640 medium supplemented with L-Glutamine and 15 mM HEPES (Gibco). The digested tissues were then pestled through 70 μm cell strainers (ThermoFisher Scientific) with RPMI-1640 (supplemented as before), and then washed twice with 1X DPBS. A Percoll (GE Healthcare) density gradient centrifugation was used to enrich the leukocyte fraction and remove blood and debris from the muscle samples and centrifuged at 2100 x g for 30 min with the lowest acceleration and no brake. Cells were washed 1X DPBS, stained with a viability live/dead amine reactive dye, washed with 1X DPS, blocked with anti-mouse TruStain FcX (BioLegend), and surface stained. Flow cytometry was performed using an Attune NxT Flow Cytometer (ThermoFisher Scientific) with a Violet, Blue, Green, and Red laser configuration. All subsequent analyses were performed on Live singlet cells using Windows based FlowJo TM software v10 (Benton Dickinson), license supplemented courtesy of the Johns Hopkins Bloomberg Flow Cytometry and Immunology Core.
8
Multiparameter Flow Cytometry Analysis of Hematopoietic Reconstitution
9
Multiparameter Flow Cytometry Analysis of Hematopoietic Reconstitution
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Gating strategies can be found in Supplemental Information (Figures S7 and S8 ). For analysis of mixed chimerism, cells were first stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (ThermoFisher Scientific) and blocked with TruStain FcX anti-mouse (Biolegend) for 10 min on ice in Cell Stain Buffer (Biolegend). Antibodies used for staining from Biolegend were as follows: CD45.1 PerCp-Cy5.5 (A20), CD45.2 Pacific Blue (104), CD3 AF488 (17A2), CD4 PE (RM4–4), CD11b BV605 (M1/70), CD19 PE-Cy7 (6D5), CD49b APC (DX5), CD25 PE-Cy5 (PC61), CD8a PE (53–6.7), Ter-119 PE, CD11b PE (M1/70), Gr-1 PE (RB6–8C5), CD3 PE (17A2), B220 PE (RA3–6B2), CD317 PE (927), CD172a APC (P84), CD11c AF700 (N418), CD304 PE (3E12), FOXP3 AF647 (150D), Helios AF488 (22F6), B220 FITC (RA3–6B2), CD8a BV510 (53–6.7). eBioscience antibodies used were as follows: CD117 APC (2B8), Sca-1 Pe-Cy7 (D7). Staining of intracellular markers was conducted with Biolegend True-Nuclear Transcription Factor Buffer Set as per manufacturer’s instructions. For live cell sorting, propidium iodide (MilliporeSigma) was used to determine viability. Cells were analyzed and/or sorted with a BD FACSAria II. Data were analyzed using FlowJo (10.7).
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