The level of apoptosis of the cells was analyzed by flow cytometry. Tumor cells were transfected with PCK1 OE lentivirus, miR-4477b inhibitor, or the corresponding NCs. After 48 h of incubation, cells were collected and stained with Annexin V/FITC kit (Beyotime, China). Finally, the cells were analyzed by a flow cytometry machine (BD, USA).
Flow cytometry machine
Manufactured by BD
Sourced in United States
A flow cytometry machine is an instrument used to detect and measure physical and chemical characteristics of particles, such as cells, in a fluid as they pass through a beam of light. It functions by analyzing the light scattering and fluorescence properties of these particles.
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Lab products found in correlation
Annexin 5 fitc kit, by Beyotime (1 mentions)
Annexin 5 fitc propidium iodide apoptosis detection kit, by BD (1 mentions)
Trypsin, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Annexin 5 fitc pi detection kit, by Sangon (1 mentions)
Cellquest pro software, by BD (1 mentions)
Annexin 5 kit, by BD (1 mentions)
7 protocols using flow cytometry machine
1
Quantifying Apoptosis in Tumor Cells
2
Annexin V-FITC/PI Apoptosis Assay
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Cell apoptosis was assessed by an Annexin V-FITC/Propidium Iodide (PI) apoptosis detection kit (BD Pharmingen, U.S.A.). Briefly, NPCs were trypsinised with 0.25% trypsin (Sigma, St. Louis, MO, U.S.A.) and washed twice with phosphate buffer solution (PBS, Solarbio, China) after centrifugation at 4°C. The cells were resuspended in 200 μl of binding buffer and mixed with 10 μl of Annexin V-FITC solution in the dark at room temperature for 15 min. Then 10 μl of PI and 300 μl of binding buffer were added for another 5 min. Thereafter, NPCs were subjected to a flow cytometry machine (BD Biosciences, San Jose, CA, U.S.A.) to analyse the apoptosis ratio. Apoptotic cells, stained positive for Annexin V-FITC, negative for PI, and double positive, were counted. Data were represented as a percentage of the total cell count.
3
Apoptosis Quantification by Annexin V/PI Assay
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Cell apoptosis was measured by using fluorescein isothiocyanate-labeled Annexin V/propidium iodide (Annexin V-FITC/PI) detection kit (Sangon Biotech, Shanghai, China). At 12 h post-transfection, cells were collected and washed with ice-cold PBS. Subsequently, 1× binding buffer was used to resuspend the cells and 5 µL of annexin V and PI was utilized to stain the apoptotic cells in the dark, according to the manufacturer’s protocol. Finally, the proportion of apoptotic cells was counted via a flow cytometry machine (BD Biosciences, San Jose, CA, USA).
4
Flow Cytometry Analysis of PD-L1 and HLA-ABC
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Flow cytometry was performed as previously described [29 ]. Briefly, cell suspensions were washed with PBS and then directly incubated with indicated fluorescence‐labeled antibodies (such as anti‐PD‐L1 or HLA‐ABC antibodies) or isotype controls for 1 h at 4°C. Subsequently, the cells were washed and resuspended with PBS, and then fluorescence data were collected on a flow cytometry machine (BD, San Diego, CA, USA). The data were analyzed using the FlowJo 7.6.1 software (TreeStar, Ashland, OR, USA). PE‐conjugated PD‐L1 antibody (#393608) and PE‐Cy5.5‐conjugated HLA‐ABC antibody (#311408) were purchased from Biolegend (San Diego, CA, USA).
5
Characterization of hASCs surface markers
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The cells stimulated by LIPUS were digested with 0.25% trypsin–EDTA and collected. The expressions of hASCs surface markers CD105-PE, CD73-PE, HLA-ABC-PE, CD14-FITC, CD34-PE, and CD45-PE were determined by flow cytometry. The cells were removed and washed with D-PBS buffer twice. 3 × 105 /mL of hASCs were resuspended in the D-PBS, and then centrifuged at 3,000 rpm for 5 min; after the supernatant was removed, the fluorescent monoclonal antibody was added and incubated at room temperature in the dark for 30 min and detected in the flow cytometry machine (Becton–Dickinson, USA).
6
Flow Cytometric Analysis of T-Cell Subsets
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The splenocytes (106 cells) were isolated from 3 mice from each group before and after challenge infection, washed twice with PBS (pH 7.4), and resuspended in PBS containing 10% fetal bovine serum (FBS). The splenocytes were incubated with anti-mouse CD16/CD32 antibody and monoclonal antibody of fluorescein isothiocyanate (FITC)-labeled CD4, phycoerythrin (PE)-labeled CD8, and allophycocyanin (APC)-labeled CD3 (eBioscience, San Diego, CA, USA) in a final reaction volume of 20 μl for 30 min on ice in the dark. The cells were washed once with PBS and fixed with 500 μl of 5% formaldehyde. After washing with PBS twice, the cells were resuspended in 1 ml PBS and loaded on a flow cytometry machine (Becton Dickinson, Franklin Lakes, NJ, USA). Cells were selected using the CD3 signal as gate R1 and the proportion of CD4+ or CD8+ subpopulation in CD3+ T cells was analyzed. Data were analyzed using BD CellQuestTM Pro software.
7
Annexin V Apoptosis Detection Assay
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The Annexin V/FITC assay was performed using Annexin V Kit (BD Pharmigen, USA) in order to analyse the potential of B1 AMCE in causing apoptosis. The cells were seeded in a 6-well plate at a concentration of 2.4 × 105 cells/mL and incubated overnight. On the next day, the seeded cells were treated with the IC50 value of Annona muricata crude extract (AMCE) and incubated for 48 and 72 h. The cells were harvested according to the incubation time point and the resulting pellets were resuspended in the binding buffer provided. Five microliter of FITC Annexin V and 5 uL of PI were added to stain the cells suspension and allowed to stand in a dark place at room temperature for 15 min. Afterwards, the stained cells were analysed by flow cytometry machine (Becton Dickinson, USA).
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