For cell cycle analysis, glioma cells (5 days) were washed twice with PBS and fixed in 75% cold ethanol at 4°C overnight. Fixed cells were washed with cooled PBS and stained using the cell cycle assay kit (Bestbio, BB-4104). The experiment was repeated three times. For proliferation and apoptosis analysis, Caspase-3, Ki-67 and cleaved poly (ADP-ribose) polymerase (PARP) were labeled with conjugated monoclonal antibodies (BD Biosciences) after U87 cell permeabilization. For CD133 expression analysis, cells were blocked by FcR blocking reagent (Miltenyi, 130-059-901) and then incubated with CD133/1-APC (Miltenyi, 130-090-826) at 4°C for 30 min. Cells were washed twice and resuspended with cold PBS. 7-AAD (BD Biosciences, 51-68981E) was added to cell suspensions to identify dead cells. Mouse IgG1-APC (Miltenyi, 130-092-214) was used as the isotype control. All analyses were performed on a FACS Calibur analyzer (BD Biosciences) using FlowJo software (Tree Star).
Mouse igg1 apc
Manufactured by Miltenyi Biotec
Sourced in United Kingdom, Germany
Mouse IgG1-APC is a fluorescently labeled antibody that can be used for flow cytometric applications. It targets the IgG1 isotype of mouse immunoglobulins and is conjugated to the fluorescent dye Allophycocyanin (APC).
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 3, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Facscalibur analyzer, by BD (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
7 aad, by BD (1 mentions)
Cell cycle assay kit, by BestBio (1 mentions)
Cd133 1 apc, by Miltenyi Biotec (1 mentions)
Mouse igg1 fitc, by BD (1 mentions)
Kdr pe fab357p, by R&D Systems (1 mentions)
Cyan adp flow cytometer, by Beckman Coulter (1 mentions)
Cd271 apc, by Miltenyi Biotec (1 mentions)
Cd146 vioblue, by Miltenyi Biotec (1 mentions)
Mouse igg1 pe, by Miltenyi Biotec (1 mentions)
Cd34 pe vio770, by Miltenyi Biotec (1 mentions)
Cd45 percp, by Miltenyi Biotec (1 mentions)
Mouse igg1 fitc, by Miltenyi Biotec (1 mentions)
Cd90 fitc, by Miltenyi Biotec (1 mentions)
Facscalibur system, by BD (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Dojindo Laboratories (1 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
9 protocols using mouse igg1 apc
1
Comprehensive Cell Cycle and Apoptosis Analysis
2
Quantifying CD146 Expression in AF Cells
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TGF-β1-treated and -non-treated AF cells were harvested by trypsinisation. Single-cell suspension was prepared at a cell density of 1 × 105 cells per 100 μL staining buffer (PBS with 0.2 % BSA and 1 mM EDTA). Cell suspensions were incubated at 4 °C in the dark for 30 min with 10 μL fluorescence-conjugated mouse monoclonal anti-human CD146 antibody (CD146-APC, human, Miltenyi Biotec) or 10 μL mouse IgG1-APC (isotype control, Miltenyi Biotech). After incubation and washing, DAPI at a final concentration of 0.1 μg/mL was added for live/dead staining. Flow cytometric analysis was performed on a FACS AriaIII (BD Biosciences) and at least 30,000 events per sample were recorded. Data analysis was performed using BD FACSDiva software. A gating strategy was used to exclude dead cells and cell doublets.
3
Flow Cytometric Characterization of Adherent Cells
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At day 3 or 7 of differentiation, adherent cells were harvested using 0.25% TrypleE with EDTA and made into a single-cell suspension in PBS with 0.2% BSA; then all cells were analyzed using flow cytometry directly without purification. Mouse Anti-Human APJ APC-conjugated Antibody (R&D, Catalog Number: FAB8561A) was used as a ratio of 1：50, Mouse IgG1 (FITC, Material Number: 551954, BD Pharmingen), Mouse Anti-human CD31 (CD31-FITC, Material Number: 555824, BD Pharmingen), Mouse IgG1-APC (Order no: 130092214, Miltenyi Biotec), Mouse Anti-human CD34 (CD34-APC, Material Number: 560940, BD Pharmingen), Mouse Anti-human CD43 (CD43-APC, Material Number: 560198, BD Pharmingen), Mouse Anti-Hamster IgG PE-conjugated Antibody (Catalog Number: F0120, R&D system), Mouse Anti-human KDR (KDR-PE, FAB357P, R&D) and Mouse Anti-Human NRP-1 (NRP-1-PE, Material Number: 565951, BD) antibodies were used at a ratio of 1：20. Single-cell suspensions were subse-quently incubated with antibody or antibodies at 4℃ for about 40 mins. Flow cytometric detection of the cell surface antigens were performed on a BD AccuriTM C6 Plus personal flow cytometer (Becton Dickinson). Compensation was set by single-positive controls.
4
Surface Marker Analysis of Sorted Cells
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Cells from the sorting procedure were centrifuged at 300g for 10 min and pellets resuspended in the appropriate fluorescent antibodies or isotype control antibodies at a concentration of 1:11 in MACS buffer. Cells were incubated in up to three antibodies for 10 min at 4 °C. After a final wash step, cells were resuspended in MACS buffer and transported directly for flow cytometry and analysed for surface marker expression using a Cyan ADP flow cytometer (Beckman Coulter, High Wycombe, UK) at the Faculty of Biology, Medicine and Health Core Facility, University of Manchester. Compensation was carried out using a bead kit (Miltenyi Biotec, Surrey, UK, 130-097-900). All antibodies and isotype controls used were obtained from Miltenyi Biotech (Surrey, UK) and are as follows with product codes: CD29-PE (130-101-275), CD34-PE-Vio770 (130-100-844), CD45-PerCP (130-098-145), CD90-FITC (130-097-930), CD146-VioBlue (130-099-678), CD271-APC (130-091-884), Mouse IgG1-PE (130-098-845), Mouse IgG2a-PE-Vio770 (130-098-564), Mouse IgG2a-PerCP (130-099-190), Mouse IgG1-FITC (130-098-847), Mouse IgG1-VioBlue (130-099-756), and Mouse IgG1-APC (130-098-846). Data were analysed using FlowJo v10 (FlowJo LLC, Ashland, OR, USA).
5
Cell Surface Marker and Apoptosis Analysis
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The expression of the cell surface molecule was determined by staining cells with CD44‐APC, human (clone: DB105) and CD133/2(293C3)‐APC, human (clone: 293C3) (Miltenyi Biotec GmbH, Germany) according to the instruction book. Mouse IgG1‐APC and Mouse IgG2b‐APC (Miltenyi Biotec GmbH, Germany) were used as isotype controls. Cellular apoptosis was detected by flow cytometry using the Annexin V FITC Apoptosis Detection Kit (DOJINDO, Japan) according to the manufacturer's instructions. In addition, apoptosis was assessed by microscopic imaging (Nikon, Japan) with original magnification of 20×. Cell cycle progression was detected by flow cytometry using the Cell Cycle and Apoptosis Analysis Kit (Beyotime, Shanghai, China). Data were acquired and analyzed using FACSCalibur system (BD Bioscience, Piscataway, NJ, USA).
6
Expression and Localization of VASP in Jurkat T-cells
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1x107 stably transduced Jurkat T-cells (Jurkat scramble or VASP-KO, see Knockout of VASP by CRISPR/Cas9) were transfected with Tax expression plasmids pEF-1α-Tax, p8-HA, VASP-FLAG (50 μg each) or both p8-HA and VASP-FLAG. Cells transfected with empty vectors pME and pEF-1α served as control. All samples were replenished with the respective empty vectors to 100 μg. At 48 h post transfection, cells were washed in PBS with 0.5% FCS and 2 mM EDTA (wash buffer) and stained without permeabilization in wash buffer using anti-HA-APC or the respective isotype-matched control antibody mouse IgG1-APC (both Miltenyi Biotec, 1:40, 10 min, 20°C). After another two washing steps, cells were analyzed with the BD LSRII flow cytometer.
7
Characterization of Cardiac Progenitor Cells
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CDCs were dissociated to single cell suspension by incubation with 0.05% Trypsin-EDTA and resuspended in DMEM/F12 with HEPES (Gibco) supplemented with 10% FBS (FACS Staining Medium). They were incubated with conjugated antibody at 1:10 dilution for 1 h at 4°C prior flow cytometry analysis (BD FACSCalibur). Antibodies used include CD105 (mouse monoclonal, Abcam), CD90 (mouse monocolonal, BioLegend), CD45 (mouse monocolonal, AbD Serotec), mouse IgG1-FITC (R&D Systems), and mouse IgG1-APC (Miltenyi Biotec). For immunocytochemistry, cells were fixed with 4% paraformaldehyde and immunostained with primary antibodies GATA4 (goat polyclonal, R&D Systems), Nkx2.5 (goat polyclonal, R&D Systems), and cardiac troponin I (mouse monoclonal, Abcam). Primary antibodies were used at a 1:100 dilution. Alexa Fluor conjugated 488 or 555 donkey anti-mouse, goat or rabbit secondary antibodies (Molecular Probes) were added at a dilution of 1:500 for 1 hour at room temperature. Fixed cultures were counterstained with DAPI (Sigma). Images were obtained using a Zeiss Axiovert 200 inverted fluorescence microscope.
8
Multiparameter Flow Cytometry Analysis
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A single cell suspension was obtained by grinding and filtering tissue, and the A549 cell suspension was digested with trypsin. All cells were resuspended in wash buffer (PBS containing 0.1% BSA) and stained for surface markers using FITC Mouse Anti-Human CD2 (BD Biosciences, 555326), FITC Mouse Anti-Human CD4 (BD Biosciences, 550628), FITC Mouse IgG1, κ Isotype Control (BD Biosciences, 555748), CD133/1 Antibody, anti-human, APC (Miltenyi Biotec, 130-113-668), and isotype control antibodies, mouse IgG1, APC (Miltenyi Biotec, 130-113-196). FCM data were acquired with a FACSCalibur (BD Biosciences). All used antibodies were list in Table S1 .
9
Quantifying CD146 Expression in AF Cells
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