The amount of LC3-II, which is recognized as a marker that is altered during autophagic flux, was evaluated by indirect immunofluorescent labeling for GFP-LC3-II and subsequent flow cytometry analysis. The cells were fixed and permeabilized with the Fixation/Permeabilization Solution Kit (BD Biosciences Pharmingen, San Diego, USA) for 20 min at 4 °C. After rinsing twice, the cells were incubated with an LC3B-specific antibody (rabbit polyclonal, ProteinTech, Illinois, USA, # 18725-1-AP) at a 1:200 dilution at 4 °C overnight. Then, the cells were rinsed three times to remove additional primary antibody before incubation at room temperature in the dark for 2 h with goat anti-rabbit DyLight 488 IgG (1:200, AntiProtech Inc., California, USA). The labeled cells were analyzed using a Beckman Coulter Epics XL cytometer (Beckman Coulter).
Epics xl cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Epics XL cytometer is a flow cytometry instrument designed for analysis of cell populations. It utilizes laser technology to detect and measure multiple parameters of individual cells or particles suspended in a fluid stream.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (7 mentions)
Dnase free rnase a, by Merck Group (5 mentions)
Cd105, by BD (2 mentions)
Anti mouse cd16 32, by BioLegend (2 mentions)
Winlist analysis software, by Verity Software House (2 mentions)
Cd14 pe, by Thermo Fisher Scientific (2 mentions)
Cd11b pe, by Thermo Fisher Scientific (2 mentions)
Cyan adp dako, by Agilent Technologies (2 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Anti cd16 cd32 antibody, by BioLegend (1 mentions)
Rnase a, by Merck Group (1 mentions)
Cd146, by BD (1 mentions)
Cd105, by Thermo Fisher Scientific (1 mentions)
Pe anti mouse cd86, by BioLegend (1 mentions)
Apc anti mouse cd206, by BioLegend (1 mentions)
Edu assay kit, by Beyotime (1 mentions)
Annexin 5 and pi, by Roche (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Dcfh2 da, by Merck Group (1 mentions)
Kaluza analysis software, by Beckman Coulter (1 mentions)
35 protocols using epics xl cytometer
1
Quantifying Autophagic Flux via GFP-LC3-II
2
Analyzing RAW264.7 Cell Surface Markers
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The cell surface markers of RAW264.7 cells (before or following coculture with YMSCs and AMSCs (with or without IFNγ stimulation)) were analyzed by flow cytometry. Briefly, cells were trypsinized, washed twice with PBS (HyClone) and divided into 1 × 106 cells/tube. For blocking of Fc receptors in the flow cytometric analysis, the cells were preincubated with purified anti-CD16/CD32 antibody (reached a concentration of 1%; Biolegend, San Diego, CA, USA) for 10 min on ice. Then, antibodies for cell markers were added to the indicated tubes (antibodies against mouse CD11b and F4/80 were used to stain untreated RAW264.7 cells); RAW264.7 cells (and the control cells) were stained separately with antibodies against mouse CD86 and CD206 following coculture and mixed into the final antibody concentration (antibodies against mouse CD11b, F4/80 and CD206 reached a concentration of 1%; antibodies against mouse CD86 reached a concentration of 6.25‰; all from Biolegend). Cells not treated with antibodies were defined as the blank controls. Cell suspension with antibodies was incubated on ice for 30 min in the dark and washed twice with PBS. The positively stained cells were detected with a Beckman Coulter Epics XL cytometer (Beckman Coulter, Fullerton, CA, USA).
3
Cell Cycle Analysis by Flow Cytometry
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Cell cycle distribution was determined by flow cytometry (Becton–Dickinson and Beckman– Coulter). After 48 h incubation with compounds, cells were harvested and fixed in 70% ethanol at 4 °C for 24 h. Then, cells were incubated with RNaseA (100 mg/l) at 37 °C for 30 min and stained with propidium iodide (10 mg/l) (Sigma-Aldrich). Samples were analyzed for DNA content using a Coulter EPICS-XL Cytometer.
4
Platelet VASP Phosphorylation Assay
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The VASP phosphorylation test was performed more than 1 h after blood collection by an experienced technician using Platelet VASP kits (Becton Dickinson, USA) according to the instruction manual [10 (link)]. Briefly, blood samples were mixed in vitro with adenosine diphosphate (ADP) and/or prostaglandin E1 (PGE1). Each blood sample was incubated with a 16C2FITC antibody, followed by a goat anti-mouse fluorescence staining in isothiocyanate polyclonal reagent. Flow cytometric monitoring was conducted with a Coulter EPICS XL cytometer (CA, U.S.A). The platelet group was identified on its side scatter and forward distributions. Every 3000 platelet events were gated and analysed for mean fluorescence intensity (MFI) using flow cytometry. The MFI corresponding to each experimental situation (ADP and ADP + PGE1) was determined to calculate a ratio directly related to the VASP phosphorylation value. The ratio, [(MFIPGE1-MFIADP + PGE1)/MFIPGE1] × 100%, which indicates a percentage of platelet reactivity, is expressed as a PRI corresponding to a ratio of activated platelets versus resting platelets.
5
Phenotypic Analysis of PDLSCs
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The single-cell suspension of 5×105 PDLSCs (P5) was incubated with antibodies for human Stro-1 (PE), CD146 (PE), CD105 (PE), CD29 (FITC), CD34 (PE) and CD45 (APC) (BD Bioscience, USA) at 4°C without light. The samples were detected by flow-cytometry using a Beckman Coulter Epics XL cytometer (Beckman Coulter, USA) to identify stem cell surface markers.
6
Immunophenotyping of Bone Marrow Stem Cells
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The expression of mesenchymal and non-mesenchymal stem cell-associated surface markers that are the characters of the immunophenotype of BMSCs were measured by flow cytometric analysis at early passages (P3). Briefly, approximately 5 × 105 liberated adherent BMSCs were detached from the culture flasks and suspended in PBS containing 3% FBS in different EP tubes, and the following antibodies were incubated for 20 min at room temperature in the dark: CD4, CD44, CD45, CD90, CD105 (eBioscience, U.S.A.). A negative panel of the following surface antigens was incubated simultaneously as a group in the same sample. Cell suspension without antibodies served as a control group to determine background fluorescence. After 1 h, the cells were washed with PBS containing 3% FBS for three times and 300 ml of the suspension was added into the testing tubes, equipped with laser emission at 488, 633 and 407 nm. The FITC and PE channels were used to detect the emission of conjugated surface antigens [23 (link)]. Finally, the samples were measured by flow cytometric analysis using a Beckman Coulter Epics XL cytometer (Beckman Coulter, Fullerton, CA, U.S.A.).
7
Immunophenotyping of M0, M1 and M2 Macrophages
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After stimulation for 24 hrs, the immunophenotype of RAW 264.7 cells was evaluated by flow cytometry, as described previously 31 . Briefly, 1 × 106 M0‐unpolarized, M1‐polarized or M2‐polarized Mφs were trypsinized and washed twice with cold PBS. The cell suspension was then divided into sterile Eppendorf tubes and blocked with 2% anti‐mouse CD16/32 (Biolegend, San Diego, CA, USA) on ice for 10 min. The cells were then washed twice and incubated with the following antibodies for half an hour in the dark: PE anti‐mouse CD86 and APC anti‐mouse CD206 (both from Biolegend). Cells that were not pretreated with any antibody were used as blank controls. The cells were washed twice to remove excess antibodies, resuspended in 400 μl PBS containing 3% FBS and analysed with a Beckman Coulter Epics XL cytometer (Beckman Coulter, Fullerton, CA, USA).
8
Multiparametric Analysis of Cell Populations
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5 × 105 cells were incubated with anti-human CD34, CD45, CD90, and CD105 antibodies labelled with PE and anti-human CD73 antibodies labelled with FITC (all from eBioscience, San Diego, CA, USA) for 30 min, respectively, at room temperature. Primary macrophages or Raw264.7 cells were stained with CD206 labelled with PE for 30 min at room temperature. T cells were stained with CD4, CD8, CD25, and Foxp3. All the surface markers were analyzed by flow cytometry with a Beckman Coulter Epics XL cytometer (Beckman Coulter, USA). Apoptosis was determined by Annexin V and PI (Roche) staining according to the manufacturer's protocol. Proliferation was determined by the EdU Assay Kit (Beyotime Biotechnology, China).
9
Apoptosis Assay for C4-2B Cells
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Assay performed according to the manufacturer's instructions (Annexin V-FITC Apoptosis Detection Kit, Phoenix Flow Systems, San Diego, CA, USA). Briefly, C4-2B cells were serum-starved overnight, treated with 4 μM SIM and/or 2 mM MET for 48−96 h, trypsinized, washed with 1 × PBS, and 1 × 106 cells incubated and stained with AV and PI, and analyzed using an Epics XL cytometer (Beckman Coulter), EXPO32 acquisition software, and WinList analysis software (version 7, Verity Software House Inc.) alongside control samples.
10
NBT Reduction Assay and Immunophenotyping
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