The FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA) was used to detect apoptosis rate of cells. The collected fresh cell pellets were washed using cold PBS and then suspended by 1 × binding buffer. Next, 5 ul of Annexin V and 5 ul of PI was added into 300 ul of 1 × binding buffer contained around 1 × 105 cells. Cells were gently mixed and then incubated for 15 min at room temperature in the dark. Then cells were analyzed on the FACS Calibur System (Beckman Coulter). The PE Mouse Anti-Human PD-L1 (Cat 329706; BioLegend, Inc.), APC Mouse Anti-Human PD-L2 (Cat 345507; BioLegend, Inc.), and PerCP/Cyanine5.5 Mouse Anti-Human B7-H3 (Cat 351009; BioLegend, Inc.) were used to detect PD-L1, PD-L2, and B7-H3 in MCF-7 and MDA-MB-213 cells. First, fresh cell pellets were collected and washed with PBS, then were suspended in 300 ul of PBS containing 1‰ BSA and incubated with corresponding antibody for 30 min on ice in the dark. Finally, staining cells were washed and then analyzed on FACS Calibur System (Beckman Coulter). FlowJo (ver. 10.0) was used for data acquisition and analysis.
Facscalibur system
Manufactured by Beckman Coulter
Sourced in United States
The FACSCalibur system is a flow cytometry instrument designed for multi-parameter analysis of cells and particles. It features dual-laser excitation and four-color detection capabilities, enabling the simultaneous measurement of multiple cellular properties. The system is capable of analyzing a wide range of biological samples, including cells, microorganisms, and sub-cellular structures.
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Lab products found in correlation
Cd73 apc, by Thermo Fisher Scientific (4 mentions)
Cd105 pe, by Thermo Fisher Scientific (4 mentions)
Cd90 fitc, by Thermo Fisher Scientific (4 mentions)
Hla dr pe cy5, by BioLegend (4 mentions)
Cd45 fitc, by BioLegend (4 mentions)
Cell cycle analysis kit, by Beyotime (3 mentions)
Cd34 pe, by Thermo Fisher Scientific (3 mentions)
Annexin 5 fitc and pi, by Beyotime (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
Cellquest software version 3, by BD (1 mentions)
Fc500mcl, by Beckman Coulter (1 mentions)
Hla dr, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Pi rnase mixture, by Beyotime (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
Annexin 5 fluos staining solution, by Roche (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Pi rnase staining buffer, by BD (1 mentions)
32 protocols using facscalibur system
1
Apoptosis and Immune Checkpoint Analysis
2
Annexin V-FITC Apoptosis Assay
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According to the protocol of the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China), cells were transfected with miR-NC, miR-486-5p, si-NC or si-EVI5. After 48 h, cells were harvested, washed with cold PBS, and resuspended in binding buffer containing Annexin V/FITC and PI (Beyotime). Stained cells were then detected using the FACS Calibur system (Beckman Coulter).
3
Isolating and Analyzing PBMs from Blood
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Blood samples (10 ml) were drawn using a needle and syringe from the peripheral vein of all patients, after which PBMs were isolated from the heparinized (Well-Biology) blood samples by Ficoll-Hypaque density gradient centrifugation at 150 × g and 4°C for 20 min. Cells were then incubated at 4°C for 45 min in the dark with monoclonal PE-conjugated mouse anti-CD14 (1:10; sc-52457; Santa Cruz Biotechnology, Inc.) monoclonal FITC-conjugated mouse anti-HLA-DR (1:10; sc-33718; Santa Cruz Biotechnology, Inc.) and monoclonal PerCP-conjugated mouse anti-CD80 antibodies (1:10, sc-73382; Santa Cruz Biotechnology, Inc.). Samples were assayed using a FACSCalibur system (Beckman Coulter, Inc., Brea, CA, USA) and analysis was performed using CellQuest software, version 3.0 (BD Biosciences, Franklin Lakes, NJ, USA).
4
Phenotypic Characterization of Cultured MenSCs
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Surface markers of cultured MenSCs were analyzed using a flow cytometer (FC500MCL, Beckman Coulter, United States). The third and fifth passages of MenSCs ( 5 x 105 cells/100 μL) were incubated directly with phycoerythrin (PE) or allophycocyanin (APC)-conjugated mouse monoclonal antibodies against human CD9, CD29, CD41a, CD44, CD59, CD 73, CD90, CD105, CD14, CD34, CD45, CD117, and human leukocyte antigen (HLA)-DR (BD Biosciences, CA, United States) for 30 min in the dark at 4 °C, followed by washing and resuspension in PBS twice. Flow cytometry was conducted using a FACSCalibur system (FC500, Beckman Coulter, United States). Data were analyzed using FlowJo Version 10.05.
5
Flow Cytometry Analysis of Endothelial Progenitor Cells
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EPCs in peripheral and renal vein blood were identified by flow cytometry using double staining as depicted in our recent report [39 (link)] using a fluorescence-activated cell sorter (FACSCalibur™ system; Beckman Coulter Inc, Brea, CA). Every analysis included 300,000 cells per sample. The assays for circulatory and coronary sinus EPCs in each sample were performed in duplicate and mean levels were reported. Intra-assay variability based on repeated measurement of the same blood sample was low with a mean CV of 3.9% study subjects.
One blood sample was drawn at 8:00 a.m. prior to G-CSF injection and the other was collected following G-CSF treatment for flow cytometric analysis. In addition, to elucidate the serial changes in the levels of EPC in renal vein, a series of blood samples were drawn from the renal vein at 0 minute prior to CD34+ cell transfusion and at 5, 10, and 30 minutes after CD34+ cell transfusion for flow cytometric analysis.
One blood sample was drawn at 8:00 a.m. prior to G-CSF injection and the other was collected following G-CSF treatment for flow cytometric analysis. In addition, to elucidate the serial changes in the levels of EPC in renal vein, a series of blood samples were drawn from the renal vein at 0 minute prior to CD34+ cell transfusion and at 5, 10, and 30 minutes after CD34+ cell transfusion for flow cytometric analysis.
6
DNA Content Analysis by Flow Cytometry
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After different oxygen treatment, the radioresistant cells were harvested and fixed in 70% ice cold ethanol and followed by RNase A treatment, stained with 50 lg/mL of propidium iodide for the DNA content analysis by flow cytometry on a FACS Calibur system (EPICS ALTRA, Beckman Coulter, Fullerton, CA). The data were processed by FlowJo FACS analysis software (Tree Star, Ashland, OR) [16 (link)].
7
Apoptosis Evaluation by Flow Cytometry
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For evaluation of apoptosis, cells were treated with gradient concentrations of GW-8510 for 24 h, and then labeled with the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, USA) following the manufacturer’s protocol. Thereafter, cells were analyzed immediately using a flow cytometry FACS Calibur System (Beckman Coulter).
8
Cell Cycle Analysis via Flow Cytometry
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According to the protocol of the Cell Cycle Analysis Kit (Beyotime, Shanghai, China), cells were cultured in 6-well plates and transfected with negative control miRNA (miR-NC), miR-486-5p, si-NC or si-EVI5 for 48 h. Cells were then harvested, washed with cold PBS, fixed with 70% ethanol at 4 °C for 24 h, washed with cold PBS again and stained with a propidium iodide (PI)/RNase A mixture. Next, cells were incubated in the dark at 37 °C for 30 min and analysed using a fluorescence-activated cell sorting (FACS) Calibur system (Beckman Coulter, Brea, CA, USA).
9
Phenotypic Analysis of hBMSCs
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Culture-expanded cells (passages 3–6) were washed with phosphate-buffered saline (PBS) containing 0.5% (w/v) bovine serum albumin (BSA), their concentration adjusted to 1×106 cells/100 µl, and phenotypic analyses performed via flow cytometry. The hBMSCs were blocked with 1% BSA and incubated with phycoerythrin- or fluorescein isothiocyanate-conjugated mouse monoclonal antibodies against human CD34 (catalog no. sc-19587; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), CD54 (catalog no. ab27582; Abcam, Cambridge, UK), CD45 (catalog no. 560976; BD Biosciences, San Jose, CA, USA), CD44 (catalog no. 560977; BD Biosciences), CD29 (catalog no. sc-59829; Santa Cruz Biotechnology, Inc.), human leukocyte antigen-antigen D related (HLA-DR; catalog no. 560944; BD Biosciences) and CD90 (catalog no. 561969; BD Biosciences) for 60 min in the dark at 4°C, at dilutions recommended by the manufacturers. Subsequently, cells were washed with PBS and fixed with 2% paraformaldehyde. Immunoglobulin isotype incubation was performed as a negative control. Flow cytometry was performed with a FACSCalibur system (FC500; Beckman Coulter, Inc., Brea, CA, USA) and analysed using FlowJo software version 7.6.5 (FlowJo, LLC, Ashland, OR, USA).
10
Identification of Endothelial Progenitor Cells
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For identification of EPCs derived from peripheral blood, flow cytometry was applied for the assessment of EPC surface markers, including sets of KDR+/CD34+/CD45−, CD133+/CD34+/CD45−, and CD34+, as described in our recent reports [6 (link), 8 (link), 11 (link)]. Briefly, mononuclear cells were isolated by density-gradient centrifugation of Ficoll-Paque Plus™ (GE Healthcare Biosciences, Uppsala, Sweden) and further incubated with fluorescent dye-conjugated mononuclear antibodies. After incubation, the mononuclear cells were fixed in 1% of paraformaldehyde for flow cytometry analysis by using a fluorescence-activated cell sorter (FACSCalibur system; Beckman Coulter, CA, USA). The assays for circulating EPCs in each sample were performed in duplicate, and the mean levels were reported.
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