For flow cytometric analysis, MSC were stained with the following antibodies: anti-mouse Sca1PE, anti-mouse CD73PE/Cy7, anti-mouse CD117APC (c-kit), anti-mouse CD45PE, anti-mouse CD44PE, anti-mouse CD95PE (Biolegend, San Diego, CA, USA), goat anti-mouse CD105 and the secondary rabbit anti-goatFITC (Invitrogen, Carlsbad, CA, USA). The antibody mix that was used to label the splenocytes contained anti-mouse CD45PE, anti-mouse CD4BV784 and anti-mouse CD8aAPCfire (Biolegend, San Diego, CA, USA). FasL was detected using anti-FasLAF647 antibody (MLF4 clone, Bio-Rad, CA, USA). For all the antibodies, we used the corresponding isotypes from the same sources. Samples were analyzed using a Cytoflex flow cytometer (Beckman Coulter, Indianapolis, IN, USA) and CytExpert software. Determination of the proliferation index of the splenocytes was done based on the carboxyfluorescein succinimidyl ester (CFSE) readings that were processed using ModFit LTTM software (Verity Software House, Topsham, ME, USA). For the CFSE analysis, the unstimulated splenocytes were used as the parent (basal fluorescence).
Pe anti mouse cd45
Manufactured by BioLegend
Sourced in United States
PE anti-mouse CD45 is a fluorescently labeled antibody that binds to the CD45 surface antigen expressed on mouse leukocytes. It can be used for the identification and enumeration of mouse immune cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Pe anti mouse sca 1, by BioLegend (3 mentions)
Pe anti mouse cd3, by BioLegend (3 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Fitc rat igg2a κ isotype control, by BioLegend (2 mentions)
Anti mouse cd4 apc cy7, by BioLegend (2 mentions)
Pe anti mouse cd29, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd44 pe, by Thermo Fisher Scientific (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (2 mentions)
Pe anti mouse cd44, by BioLegend (2 mentions)
Moflo astrios eq, by Beckman Coulter (2 mentions)
Live dead fixable near ir dead cell dye, by Thermo Fisher Scientific (2 mentions)
Percp antimouse human cd11b, by BioLegend (1 mentions)
Fitc anti mouse ly6c, by BioLegend (1 mentions)
Anti mouse cd16 cd32 fc block, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Apc anti mouse human cd44, by BioLegend (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
27 protocols using pe anti mouse cd45
1
Flow Cytometric Profiling of Murine MSCs
2
Quantifying Ly6C+ Monocytes in Mouse
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250‐300 μL of blood samples was collected for flow cytometry analysis on day 7 after AngII infusion. The following antibodies were used for staining: FITC antimouse Ly‐6C (Biolegend 128005), PerCP antimouse/human CD11b (Biolegend 101229) and PE antimouse CD45 (Biolegend 103105). Forward scatter (FSC) and side scatter (SSC) were used to gate live cells excluding red blood cells, debris and cell aggregates among total blood cells. Cells were acquired using a BD FACS Canto II (BD Biosciences) and analysed with FlowJo (Tree Star, Inc). The percentage of CD45+CD11b+ly6Chi cells among the total leucocyte population were calculated and analysed.
3
Multiparameter Flow Cytometry Assay
4
Isolation and Characterization of Intestinal Crypt Stem Cells
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The small intestine crypts were isolated at day 5 post-irradiation. The crypts were incubated in the digestive juice, which pre-warmed and contained 2 mL TRYPLE express (Gibco Life Technologies), 20 µL Y27632 (Sigma-Aldrich), 2 mL N-acetylcysteine (Sigma-Aldrich), 80 µL DNase and 2 µL Dispase Type II (Gibco Life Technologies) in 37 °C for 8 minutes. Quickly, they ended the digestive process by adding DMEM with 10% fetal bovine serum (Gibco life technology). The mixture was centrifuged at 1,200 rpm for 5 min at 4 degrees. The cells were passed through 40-µm strainers (BD Bioscience) and resuspended with ABS (~1×106 cells/100 µL). The cells were counterstained with different fluorescein antibodies, including APC anti-mouse/human CD44 (Biolegend), PE anti-mouse CD45 (Biolegend), FITC anti-mouseCD326 (Biolegend) and propidium iodide (PI, Sigma-Aldrich), according to the manufacturer’s instructions. PI(−) CD45(−) EpCAM(+) CD44(+) [marked as CD44(+)] cells are sorted using s flow cytometer (Beckman). The sorted cells were collected, and their RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). The GENEWIZ company did the RNA-seq and analysis work.
5
Microglia Immunophenotyping and Sorting
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Cells were stained with PE-anti mouse CD45 (Biolegend, Copenhagen, Denmark) and PerCP-Cy5.5-anti-mouse CD11b (for sorting of whole microglia population) or PerCP-Cy5.5-anti-mouse CD11b, biotinylated-anti-mouse CD11c (BD pharmingen) and PE-anti mouse CD45 (for analysis of CD11c+ microglia) as previously described (8 (link)), and sorted by FACSAriaTM III cell sorter (BD Biosciences, Albortslund Denmark).
6
Validating CX3CR1 Expression in Mice
7
Characterization of CXCR4 Expression in MSCs
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MSCs were detected the expression of specific surface markers, including PE anti-mouse Sca-1, PE anti-mouse CD90.2, PE anti-mouse CD29, PE anti-mouse CD44, PE anti-mouse CD45, APC anti-mouse CD31, APC anti-mouse CD34, and APC anti-mouse CD117 (Biolegend, San Diego, CA).
After MSCs were treated with 3 mM NMDA (Sigma-Aldrich) or 50 μM MK801 (Sigma-Aldrich) for 24 h, APC anti-mouse C-X-C chemokine receptor type 4 (CXCR4; Biolegend) was used for surface staining to study the effect of NMDA receptor activation on CXCR4 expression in MSCs.
MSCs were harvested and then blocked with Fc receptor blocking agent (BioLegend) for 10 min on ice. The antibodies were added and incubated for 30 min at 4°C in dark. The cells were then washed with 0.1% BSA and fixed with 1% paraformaldehyde in PBS. Cell surface staining was analyzed using a FACSCanto II (Becton Dickinson, Franklin Lakes, NJ) within 24 h of staining. FlowJo software version 7.6.1 (FlowJo, Ashland, OR) was used for data analyses.
After MSCs were treated with 3 mM NMDA (Sigma-Aldrich) or 50 μM MK801 (Sigma-Aldrich) for 24 h, APC anti-mouse C-X-C chemokine receptor type 4 (CXCR4; Biolegend) was used for surface staining to study the effect of NMDA receptor activation on CXCR4 expression in MSCs.
MSCs were harvested and then blocked with Fc receptor blocking agent (BioLegend) for 10 min on ice. The antibodies were added and incubated for 30 min at 4°C in dark. The cells were then washed with 0.1% BSA and fixed with 1% paraformaldehyde in PBS. Cell surface staining was analyzed using a FACSCanto II (Becton Dickinson, Franklin Lakes, NJ) within 24 h of staining. FlowJo software version 7.6.1 (FlowJo, Ashland, OR) was used for data analyses.
8
Bone Marrow Cell Immunophenotyping
9
Molecular Markers for Muscle Atrophy
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GEF was supplied by the Ginsentology Research Laboratory of Konkuk University (Seoul, Korea). Antibodies specific for myoblast determination protein 1 (MyoD), F-box protein (Fbx32/atrogin), muscle ring finger 1 (MuRF1), and tumor necrosis factor-α (TNF-α) were purchased from Abcam (Abcam, Cambridge, UK). Antibodies specific for myocyte enhancer factor-2 (MEF-2), cyclooxygenase-2 (COX-2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies specific for inducible nitric oxide synthase (iNOS) and interleukin 1 beta (IL-1β) were purchased from Cell Signaling Technology (Danvers, MA, USA). FITC anti-mouse CD3ε, PE anti-mouse CD45, PerCP/Cyanine5.5 anti-mouse CD4, Brilliant Violet421 anti-mouse CD8, APC anti-mouse CD62L, and Alexa Fluor700 anti-mouse CD44 were purchased from BioLegend (San Diego, CA, USA).
10
Characterization of Immune Cell Phenotypes
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FITC mouse IgG1 κ isotype control (400110), PerCP-Cy5.5 mouse IgG1 κ isotype control (400149), PE mouse IgG1 κ isotype control (400114), PerCP-Cy5.5 anti-human CD3 (300430), FITC anti-human CD8a (300906), PE anti-human CD45 (368510), PerCP-Cy5.5 anti-human IFN-γ (506528), FITC Rat IgG2a κ isotype control (400506), PerCP-Cy5.5 Rat IgG1 κ isotype control (400426), FITC anti-mouse CD3 (100204), FITC anti-mouse CD8a (100705), PE anti-mouse CD45 (103106), and PerCP-Cy5.5 anti-mouse IFN-γ (505822) were purchased from Biolegend. PE anti-human PD-L1 (557924), FITC anti-human PD-L1 (558065), FITC anti-human HLA-A2 (343303), FITC Rat IgG2b κ isotype control (400605), PE Rat IgG2a λ isotype control (400635), and PE anti-mouse PD-L1 (558091) were obtained from BD Biosciences.
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