Cell cycle/Edu experiments were performed by incubating growth phase C2C12 myoblast cultures with 10uM 5-ethynyl-2′-deoxyuridine (EdU) for 2 hours, fixing, counterstaining DNA with DAPI, and identifying Edu+ cells using a Click-IT Edu Flow Cytometry Kit (Thermo Fisher). Mitochondria were labeled in live cells using MitoTracker Green FM (Thermo Fisher) and analyzed by flow cytometry. Immune cell profiling was performed as follows: hindlimb muscles were enzymatically digested and filtered according to standard protocols (Bernet et al, 2014 ), cells were labeled with fluorescently conjugated primary antibodies according to manufacturer’s instructions, and flow cytometry performed on a MACSquant 10 analyzer. The following antibodies (Miltenyi Biotec) were used in this study: anti-CD45, anti-CD3, anti-CD49b, anti-CD11c, anti-MHC class II, anti-F4/80, anti-CD11c, and anti-GR1. The following is the gating strategy used to identify individual immune cell subtypes: NK: CD45+, CD3e−, CD49b+; Dendritic cell: CD45+, CD11c+, MHC class II+; M1 macrophage: CD45+, F4/80+, CD11b+, CD11c+; M2 macrophage: CD45+, F4/80+, CD11b+, CD11c−; Neutrophil: CD45+, CD11b+, GR1+; Pan T cells: CD45+, CD3e+; Cytotoxic T cells: CD45+, CD3e+, CD8+; Helper T cells: CD45+, CD3e+, CD4+; Regulatory T cells: CD45+, CD3e+, CD4+, CD25+. Samples were analyzed using either MACSquant software or FlowJo v10.
Anti cd45
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD45 is a monoclonal antibody that binds to the CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase expressed on the surface of all human leukocytes.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11c, by Miltenyi Biotec (3 mentions)
Ls column, by Miltenyi Biotec (3 mentions)
Anti cd3, by Miltenyi Biotec (2 mentions)
Percoll, by GE Healthcare (2 mentions)
Anti cd31, by Miltenyi Biotec (2 mentions)
Anti cd4, by Miltenyi Biotec (2 mentions)
Cyto id autophagy detection kit, by Enzo Life Sciences (2 mentions)
Anti cd44, by Abcam (2 mentions)
Facs lsr 2, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Anti cd73, by Miltenyi Biotec (2 mentions)
Hepes, by Sartorius (2 mentions)
Anti epcam, by Miltenyi Biotec (2 mentions)
Collagenase d, by Merck Group (2 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Click it edu flow cytometry kit, by Thermo Fisher Scientific (1 mentions)
Anti cd49b, by Miltenyi Biotec (1 mentions)
Anti f4 80, by Miltenyi Biotec (1 mentions)
Multimacs system, by Miltenyi Biotec (1 mentions)
Anti cd8a microbeads, by Miltenyi Biotec (1 mentions)
20 protocols using anti cd45
1
Comprehensive C2C12 Myoblast Profiling
2
Isolation of Tumor-Infiltrating Lymphocytes
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Tumours were harvested at days 8–21 and digested for 40 min at 37 °C according to Tumour Dissociation Kit protocol (Miltenyi Biotec 130–096–730). Tumours were crushed on 100 μm cell strainers and washed twice with PBS 2% FCS. Single-cell suspensions were enriched for CD45+ or CD8+ cells using the MultiMACS system (Miltenyi Biotec). In brief, cells from tumour tissues were labelled with anti-CD45 or anti-CD8a microbeads (Miltenyi Biotec 130–052–301 or 130–117–044, respectively), and then purified using the POSSEL program on MultiMACS. The positive fraction was recovered for TIL analysis by flow cytometry or ex vivo assays.
3
NK Cell Degranulation Assay for AML
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Tested items and PBMCs from patients with AML were added to each well of round-bottomed 96-well plates. After overnight coincubation with the NKCE molecules or antibodies, antihuman CD107a and CD107b antibodies (Miltenyi, 130-111-621 and 130-118-818) were added for 4 h. Cells were then washed and stained with the following mixture: viability markers, anti-CD45 (Miltenyi, 130-110-771), anti-CD33 (BD Biosciences, 564588), anti-CD56 (BD Biosciences, 557747) and anti-CD3 (BD Biosciences, 740187) antibodies. Cells were then washed, fixed and analyzed by flow cytometry. The data obtained were analyzed with Flowjo Software to assess NK cell degranulation by monitoring the expression of CD107a/b on NK cells identified as living CD45+CD33−CD56+CD3− cells.
4
Primary Liver Progenitor Cell Isolation
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Livers from 2–4-week DDC-treated mice were used for primary liver progenitor cell isolation by a discontinuous 20–50% Percoll (GE Healthcare, Hatfield, UK) gradient centrifugation, as previously described.6 (link), 7 (link) Briefly, LPCs were isolated by a modified two-step perfusion protocol, accompanied by MACS-based anti-CD45 (Miltenyi Biotec, Cologne, Germany) negative selection.
5
Isolation of Lung Epithelial Cells
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Lung tissue was cut into small pieces (1 mm3) and treated overnight at 4 °C with Trypsin/EDTA (0.25%; Gibco), penicillin/streptomycin (100 U/mL; 100 µg/mL), 2 mg/mL Collagenase A (Roche, Basel, Switzerland) and 0.04 mg/mL DNase (Sigma-Aldrich). Next, the suspension was diluted in DMEM containing 0.04 mg/mL DNase and penicillin/streptomycin, homogenized and filtered over two layers of gause filter (Sefar Nitex, Heiden, Switzerland). After centrifugation (500× g for 10 min) the pellet was resuspended in lysis buffer (15.5 mM NH4CL, 1 mM KHCO3, 0.01mM EDTA, 0.04 mg/mL DNase) for 10 min at 4 °C. The remaining pellet was resuspended in Small Airway Epithelial Cell Growth (SAGM) medium (PromoCell, Heidelberg, Germany) supplemented with penicillin/streptomycin, and kept on ice until use within 1–4 hrs. Cell suspensions were seeded into organoid cultures directly or EpCAM+ cells were isolated by anti-CD45 (#130-045-801, Miltenyi Biotec, Auburn, AL, USA) and anti-CD31 (#130-091-935, Miltenyi Biotec) coupled-microbeads and passed through LS columns (Miltenyi Biotec) according to the manufacturer’s guidelines. The flow-through was incubated with anti-EpCAM (CD326) microbeads (#130-061-101, Miltenyi Biotec) for positive selection with LS columns. Cells were resuspended in SAGM and kept on ice until use within 1–2 h.
6
Comprehensive T-cell Phenotyping by Flow Cytometry
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Cellular composition, CD4/CD8 ratio and CAR-expression was determined by flow cytometric analysis after staining with one or several of the following antibodies and reagents: anti-CD45, anti-CD4, anti-CD3, anti-CD16, anti-CD56, anti-CD19, anti-CD14, anti-CD8, anti-CD45RO, anti-CD95, anti-CD62L and 7-AAD (all Miltenyi Biotec). CAR-modified T cells were identified using the EGFRt marker and staining with an anti-EGFRt antibody (clone: C225, ImClone Systems) conjugated in-house to AlexaFluor 647. Data were acquired on a MACSQuant Analyzer 10 and analyzed using MACSQuantify software (both Miltenyi Biotec).
7
Comprehensive Flow Cytometry Analysis of EOC
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The cells were stained with Live-Dead to discriminate living cells. The following anti-human antibodies were used: anti-CD44 (1:1,000; Abcam, Cambridge, UK), anti-CD117 (non-activating AC126 clone, 1:10; Miltenyi-Biotec, Bergish Gladbach, Germany), anti-CD45 (1:10; Miltenyi-Biotec) and anti-Ki67 (1:10; BD Bioscience, Franklin Lakes, NJ, USA). All the cytofluorimetric analyses were performed using a FACS LSRII (BD); data were collected from at least 1 × 105 cells/sample and elaborated with FlowJo software (TreeStar, Ashland, OR, USA). For FACS sorting, antibody-labeled cells were separated with a MoFloAstrios Cell Sorter (Beckman Coulter, Brea, CA, USA); the purity of the sorted populations always exceeded 90%. To evaluate autophagic activity, EOC effusion cells were labeled with Cyto-ID Autophagy detection kit (Enzo Life Sciences).
For cell cycle analysis, cells were fixed with cold ethanol and then incubated for 1 h in a Dapi/RNAse solution. For cell viability analysis, cells were incubated for 15 min at 37 °C with AnnexinV/PI staining kit (Roche, Basel, Switzerland).
For cell cycle analysis, cells were fixed with cold ethanol and then incubated for 1 h in a Dapi/RNAse solution. For cell viability analysis, cells were incubated for 15 min at 37 °C with AnnexinV/PI staining kit (Roche, Basel, Switzerland).
8
Phenotypic Characterization of Cultured Cells
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CPs were stained for surface antigen expression using combinations of the following antibodies to confirm typical phenotype at P5: anti-CD31 (eBioscience, UK), anti-CD34 (eBioscience), anti-CD44 (eBioscience, UK), anti-CD45 (Miltenyi Biotec), anti-CD90 (BD Biosciences, UK), anti-CD105 (Life Technologies), and anti-CD146 (Miltenyi Biotec). After staining, fluorescence was analyzed using a FACS Canto II flow cytometer and FACS Diva software (both BD Biosciences, UK).
9
Isolation and Purification of Muscle Progenitor Cells
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MPCs were prepared from CTX-treated TA muscles of Col6a1GT/GT and LM mice according to previous report (Heredia et al., 2013 (link)). Briefly, isolated muscles were digested with 0.05% Collagenase Type 2 and 0.1% Dispase (Gibco) in DMEM for 30 min at 37 ˚C. Liberated cells were passed through Cell Strainer and collected by centrifugation. MPCs were purified by depletion of endothelial cells, leukocytes, and muscle cells using a mixture of biotin-conjugated anti-CD31, anti-CD45, and anti-α7 integrin antibodies, respectively (Miltenyi Biotec) and then streptavidin magnetic beads (Miltenyi Biotec). The flow through fraction was collected and confirmed by immunostaining for PDGFRα.
10
Flow Cytometry Analysis of MSCs
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For cytofluorimetric analysis 5 × 105 cells were incubated with conjugated monoclonal anti-CD34, anti-CD45, anti-CD73, anti-CD90 and anti-CD105 (from MACS Miltenyi Biotec). Cells were analysed on a FACSCanto flow cytometer (BD Biosciences). 7-AAD was used to exclude non-viable cells from the analysis. Data were analysed using FACSDiva Software (BD Biosciences).
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