Cell cycle and cell apoptosis analysis was carried out by flow cytometry. Cells (5×105/well) were seeded in 6-well cell culture plates and incubated in a humidified atmosphere of 5% CO2 at 37°C. Twenty-four hours later, cells were harvested with 0.5% trypsin and centrifuged. For cell cycle analysis, cells were fixed in ice-cold 70% ethanol overnight at 4°C. After being washed thrice with cold PBS, cells were resuspended in 500 µl of PBS, and 10 µl RNAseA was added for 5 min. Subsequently, 10 µl PI was added into the cell resuspension solution, and incubated for 30 min at 4°C. The cells were finally washed twice with PBS before analysis. Apoptotic analysis was performed using the Hoechst 33342/PI Apoptosis assay kit (BestBio, Shanghai, China). The samples were washed twice with ice-cold PBS and resuspended in 500 µl staining buffer, and then incubated with 5 µl of Hoechst 33342 and 5 µl of PI in the dark for 20 min at 4°C. The cells were finally washed twice with PBS before analysis. Analysis was performed on MoFlo™XDP High-Performance Cell Sorter (Beckman Coulter) and the data were analyzed with the summit v5.2 Software (Beckman Coulter).
Moflo xdp high performance cell sorter
Manufactured by Beckman Coulter
Sourced in United States
The MoFlo™ XDP High-Performance Cell Sorter is a flow cytometry instrument designed for high-speed cell sorting. It is capable of sorting cells at high rates with exceptional purity and recovery.
Automatically generated - may contain errors
Lab products found in correlation
Summit v5, by Beckman Coulter (4 mentions)
Hoechst 33342 pi apoptosis assay kit, by BestBio (2 mentions)
Annexin 5 pe 7 aad double staining kit, by BD (2 mentions)
Cd73 fitc, by BD (1 mentions)
Igg1 fitc, by BD (1 mentions)
Cd14 fitc, by BD (1 mentions)
Cd34 pe, by Thermo Fisher Scientific (1 mentions)
Cd90 pe cy5, by BD (1 mentions)
Cd45 pe cy5, by BD (1 mentions)
Igg1 pe cy5, by BD (1 mentions)
Cd105 pe, by BD (1 mentions)
Cd29 fitc, by BD (1 mentions)
Propidium iodide, by Beyotime (1 mentions)
Cell cycle staining kit, by Liankebio (1 mentions)
Accuri c6 instrument, by BD (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Hoechst 33342, by Keygen Biotech (1 mentions)
Stempro accutase, by Thermo Fisher Scientific (1 mentions)
Macs buffer, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
12 protocols using moflo xdp high performance cell sorter
1
Cell Cycle and Apoptosis Analysis
2
Characterization of HDPC Surface Markers
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Surface markers of HDPCs were identified by flow cytometry. After collecting ~1×106 cells from the third passage, HDPCs were washed twice with PBS and then suspended in PBS, before being blocked with 5% BSA for 30 min at room temperature to remove nonspecific binding. The antibodies, including CD13-PE (1:40 dilution; cat. no. 555394; BD Biosciences), CD14-FITC (1:40 dilution; cat. no. 555397; BD Biosciences), CD29-FITC (1:40 dilution; cat. no. 11-0291-82; Thermo Fisher Scientific, Inc.), CD34-PE (1:40 dilution; cat. no. 560941; BD Biosciences), CD45-PE-Cy5 (1:40 dilution; cat. no. 560974; BD Biosciences), CD73-FITC (1:10 dilution; cat. no. 561254; BD Biosciences), CD90-PE-Cy5 (1:40 dilution; cat. no. 555597; BD Biosciences) and CD105-PE (1:40 dilution; cat. no. 560839; BD Biosciences), were added and cells were incubated for 90 min at 37°C. IgG1-PE (1:40 dilution; cat. no. 556650; BD Biosciences), IgG1-FITC (1:40 dilution; cat. no. 556649; BD Biosciences) and IgG1-PE-Cy5 (1:40 dilution; cat. no. 550618; BD Biosciences) were also used as isotype controls with incubation for 90 min at 37°C. After washing with PBS, suspended HDPCs were transferred to FACS tubes and analyzed using a MOFlo™ XDP high-performance cell sorter (Beckman Coulter, Inc.) and SUMMIT version 5.0 software (Beckman Coulter, Inc.) according to the manufacturer's instructions.
3
Cell Cycle and Apoptosis Analysis
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The cell cycle and apoptosis analysis was described in our previous work (Liu et al., 2016 (link)). Briefly, cells were harvested and fixed in 70% ethanol for 24 h at -20°C, then treated with RNase A, and stained with 25 μg/ml of propidium iodide (PI) (Beyotime). For cell apoptosis analysis, the ratio of apoptotic cells was determined using an Annexin V-PE/7-AAD double staining kit (BD). Samples were all analyzed using a MoFlo XDP High-Performance Cell Sorter (Beckman Coulter, CA, United States), and the data were analyzed using Summit v.5.2 software according to the manufacturer’s protocol. At least three independent experiments were performed.
4
Cell Proliferation Assay by EdU Labeling
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Cells (1 × 105 to 3 × 106) were inoculated in 6-well plates and cultured at 37°C in a 5% CO2 incubator to the required cell density. Two hours before transfection, serum-free 1640 medium was used. The transfected cells were divided into groups according to the corresponding experiments. After 6 h, the mixed solution was sucked out and replaced with normal culture medium, and culture was continued for 48 h. Referring to the operating instructions of the EdU-647 cell proliferation test kit (Beyotime biotechnology, C0081S), the samples were processed before flow cytometry [28 (link),29 (link)]. Finally, the samples were tested by flow cytometry at a 488 nm excitation wavelength and 520 nm emission wavelength. Cells were analyzed with Moflo XDP High-Performance Cell Sorter (Beckman Coulter). Data were acquired and analyzed with Summit v5.2 software.
5
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, cells were harvested and fixed in 70% ethanol for 24 h at − 20 °C. The cells were then treated with RNase A and stained with 25 μg/ml propidium iodide (PI). For cell apoptosis analysis, cells were treated with serum starvation for 24 h, and then the ratio of apoptotic cells was determined using an Annexin V-PE/7-AAD double staining kit (BD Biosciences, MD, USA). Samples were all analyzed using a MoFlo™ XDP High-Performance Cell Sorter (Beckman Coulter, CA, USA), and the data were analyzed using Summit v.5.2 software according to the manufacturer’s protocol. At least three independent experiments were performed.
6
Cell Cycle and Apoptosis Analysis
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Cells were harvested with 0.5% trypsin and centrifuged at 1,000 × g for 5 min at room temperature. Cell cycle analysis was performed using a cell cycle staining kit (LiankeBio; cat. no. CCS012), cells were fixed in ice-cold 70% ethanol overnight at 4°C. Following being washed thrice with cold PBS, cells were suspended in 500 µl PBS, and 10 µl RNase A was added for 5 min at room temperature. Subsequently, 10 µl propidium iodide (PI; LiankeBio; cat. no. CCS012) was added into the cell resuspension solution, and incubated for 30 min at 4°C. The cells were finally washed twice with PBS prior to analysis. As for apoptotic analysis, it was performed using a Hoechst 33342/PI Apoptosis Assay kit (Shanghai BestBio Biotechnology, Shanghai, China), according to the manufacturer's protocols. The samples were washed twice with ice-cold PBS and suspended in 500 µl staining buffer, and then incubated with 5 µl Hoechst 33342 and 5 µl PI in the dark for 20 min at 4°C. The cells were finally washed twice with PBS prior to analysis. Analysis was performed on a MoFlo™ XDP High-Performance Cell Sorter and the data were analyzed with Summit v5.2 Software (both from Beckman Coulter, Inc.).
7
Identification of ALDH1A1-positive Cells
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Cell populations with high ALDH1A1 enzymatic activity were identified with the Aldefluor kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer’s protocols. Briefly, 2 × 106 cells were re-suspended in 1 mL Aldefluor buffer and 1 μL Aldefluor reagent in the presence or absence of the specific ALDH1A1 inhibitor for 30 min at 37 °C. Brightly fluorescent ALDH1A1-positive cells were detected in the green fluorescence channel, FL1, and samples treated with the specific ALDH1A1 inhibitor, DEBA, were used as the control to set the gates defining the ALDH1A1-positive region. Flow cytometry was performed using BD Accuri™ C6 instrument (BD Biosciences, San Jose, CA) as well as the data analyzing. For cell sorting, the cells were sorted a Moflo™ XDP high-performance cell sorter (Beckman Coulter, Brea, CA, USA). After sorting, the cells were washed and cultured for detection of lncRNA expression.
8
High-Performance Cell Sorting Protocol
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All cells were passaged and cultivated in 10-cm cell-culture dishes under their specific culture conditions described above. At 80–90% confluency, cells were harvested by trypsinization with 0.05% trypsin-EDTA. After washing once with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), the cells were resuspended in 5 ml of their specific culture medium and filtered through a 50-μm nylon mesh. Then, the cells were directly analyzed with a MoFlo XDP High-Performance Cell Sorter (Beckman Coulter, Fullerton, CA).
9
Isolation and Analysis of Cervical Cancer Cells
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Single-cell suspensions of cervical cancer tissues or adjacent non-cancerous tissues were prepared. Enzymatic digestion was incubated at 37°C until full digestion had occurred, with oscillations every 10–15 min prior to passing the sample through a 70-μm cell strainer. The resulting cell suspension was centrifuged (Eppendorf 5417C; Eppendorf, Engelsdorf, Germany) at 500 × g for 10 min and resuspended in saline. The cells were fixed in 500 μl paraformaldehyde 4% in Dulbecco’s phosphate-buffered saline (D-PBS) for 20 min at room temperature. Subsequent to washing in D-PBS, the cells were permeabilized with detergents (Triton X-100). The cells were washed twice with D-PBS, and the single-cell suspensions were stained and incubated at 4°C for 30 min with fluorescein isothiocyanate (FITC)-conjugated OCT-1 (Biorbyt, Cambridge, UK). Isotype controls were performed with an FITC-conjugated rabbit anti-human igG negative control (Biorbyt). All antibodies were used according to manufacturer’s instructions. The cells were washed twice and examined by FACS using a MoFlo™ XDP High-Performance Cell Sorter (Beckman Coulter, Miami, FL, USA). Data were acquired and analyzed using Summit v5.2 software (Becton Dickinson, Franklin Lakes, NJ, USA).
10
Flow Cytometric Analysis of Yeast Fluorescence
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Cells were cultured in SC medium minus leucine (−Leu) and harvested at mid‐exponential phase, washed twice with ice‐cold 10 mmol l−1 phosphate buffer (PBS, pH 7.0) and resus
pended in PBS. Samples of 2 × 105 cells were monitored through the FITC (Fluorescein isothiocyanate) channel (excitation and emission wavelengths of GFP were 488 and 507 nm, respectively) (Zhang et al.,2015 ) via flow cytometer (MoFlo™ XDP High‐Performance Cell Sorter, Beckman Coulter, USA) and analysed with the Kaluza Analysis 2.1 software. The mean fluorescence intensity (MFI) was the sum of fluorescence intensity for each single cell divided by the 2 × 105 analysed cells. Meanwhile, the cells with fluorescence intensity > 2000 a.u. were sorted out as high‐nucleic‐acid content yeast candidates.
pended in PBS. Samples of 2 × 105 cells were monitored through the FITC (Fluorescein isothiocyanate) channel (excitation and emission wavelengths of GFP were 488 and 507 nm, respectively) (Zhang et al.,
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