Flow cytometry was used to evaluate cell cycle. We collected the cells transfected with mimics and nc and added 1 mL ice-cold PBS to disperse the cells. The cells were centrifuged to remove PBS, fixed in 1 mL ice-cold 70% ethanol, and stored for 16 h at 4 °C. Then, the cells were collected and centrifuged 2 times at 1000× g for 5 min to remove ethanol. The collected cells were treated with 0.5 mL Staining Solution, 10 μL propidium iodide (PI), and 10 μL RNaseA (Yeasen, Shanghai, China) in the dark for 30 min at 37 °C. Finally, the treated cell suspension was measured by the Cytek DxP Athena flow cytometry (Cytek, Fremont, CA, USA).
Dxp athena
Manufactured by Cytek
Sourced in United States
The Dxp Athena is a flow cytometry instrument developed by Cytek. It is designed to analyze and sort cells based on their physical and fluorescent properties. The Dxp Athena utilizes multiple lasers and detectors to capture a comprehensive view of cellular characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Rnase a, by Yeasen (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
Cd11b fitc, by BioLegend (1 mentions)
F4 80 apc, by BioLegend (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
Annexin 5, by Beyotime (1 mentions)
Anti cd11c pe, by BioLegend (1 mentions)
Anti mhc 2 percpcy5, by BioLegend (1 mentions)
Anti cd11b bv605, by BioLegend (1 mentions)
Anti siglecf apc, by BioLegend (1 mentions)
Pe conjugated cd34, by BD (1 mentions)
Pe conjugated cd29, by BioLegend (1 mentions)
Pe conjugated cd90, by BD (1 mentions)
Pe conjugated cd44, by BioLegend (1 mentions)
7 protocols using dxp athena
1
Cell Cycle Analysis by Flow Cytometry
2
Apoptosis Analysis of GEM-Treated Cells
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Five thousand, six hundred thirty-seven cells were added in 6-well culture plates, followed by overnight incubation for adherence. Adherent cells were treated using 50 10− 9 м PTK7-GEMs, LIB-GEMs, GEM for 8 h and then incubated for 72 h with Complete medium, respectively, with untreated cells comprising the control. Thereafter, cells were washed with PBS, harvested, and resuspended in 5 µL of Annexin-V FITC with 195 µL binding buffer and 5 µL of 7-Aminoactinomycin D (7-AAD), according to the supplier’s guidelines (Annexin V-FITC Apoptosis Detection Kit, Beyotime Co.), and then incubated for 15 min in the dark at room temperature to analysis in flow cytometry (Cytek DxP Athena; Cytek, Fremont, CA, USA).
3
Peritoneal Immune Cell Profiling
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Single-cell suspensions from peritoneal lavage fluid were filtered by cell strainer (70 μm), followed by red blood cell lysis. Then, 1 × 106 cells were added into tubes and washed with cold PBS. And cells were incubated with mixed combinations of antibodies for 30 min at 4 °C. Information on the antibodies (BioLegend, CA, USA) used is as follows: APC-F4/80 (1:100), FITC-CD11b (1:100), PE/DAZZLE 594-CD45 (1:100), Zombie Aqua™ Fixable Viability Kit (1:500). The data were detected by DxpAthena (Cytek, USA) and analyzed by FlowJo software (TreeStar Olten, Switzerland).
4
Quantifying Apoptosis and Necrosis by Flow Cytometry
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To measure the percentage of cells undergoing apoptosis/necrosis, flow cytometry analysis using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Staining Apoptosis Detection Kit (Beyotime, China) was performed. After the relevant stimulation, cells (1 × 104 cells/well) in each group were collected, washed with PBS, and stained with 10 μL of Annexin V-FITC and 5 μL of PI for 15 min. The stained cells were detected using a Cytek Dxp Athena flow cytometer (Cytek, USA).41 (link)
5
Comprehensive Immune Cell Profiling in Murine BAL
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The bronchoalveolar lavage (BAL) fluids were obtained by injecting 1 ml PBS into the murine trachea, repeating ten times. The cells of BAL fluid were stained with anti-CD45 (APC/Cy7; BioLegend), anti-CD11c (PE; BioLegend), anti-Siglec-F (APC; BioLegend), anti-MHCII (Percp/Cy5.5; BioLegend), anti-CD11b (BV605; BioLegend), anti-Ly6G (BV421; BioLegend), and anti-CD3 (AF488; BioLegend) from light for 30 min. The cell gating strategy in BAL fluids was conducted according to the methods previously reported29 (link). The signals were obtained from a Cytek Dxp Athena flow cytometer. And the data were calculated using FlowJo software (version 10).
6
Characterization of Canine Cell Phenotypes
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The surface markers of isolated cells were analyzed by flow cytometry analysis. Briefly, the isolated cells (1 × 106 cells, passage 3) from the three dogs were, respectively, suspended in 100 μL phosphate-buffered saline (PBS) containing 10 μg/mL antibodies for PE-conjugated CD29 (303003, BioLegend, USA), PE-conjugated CD44 (103024, BioLegend, USA), PE-conjugated CD90 (561970, BD Biosciences, USA), PE-conjugated CD105 (bs-0579R-PE, Bioss, CHN), FITC-conjugated CD73 (bs-23233R-FITC, Bioss, CHN), PE-conjugated CD34 (559369, BD Biosciences, USA), and FITC-conjugated CD45 (11-5450-42, eBioscience, USA). After incubation for 30 min at 4°C, the cells were washed with PBS and then resuspended in 500 μL of PBS for analysis. As for CD11b, the isolated cells (1 × 106 cells, passage 3) were suspended in 100 μL phosphate-buffered saline (PBS) containing 10 μg/mL CD11b (MA5-16604, eBioscience, USA). After incubation for 30 min at 4°C, the cells were washed with PBS and then labeled with goat antirabbit IgG (H + L) cross-adsorbed secondary antibody, FITC (F-2765, Invitrogen), at a dilution of 1 : 500 for 1 h at room temperature. Cell fluorescence was evaluated by flow cytometry using a DxP Athena™ flow cytometry system (Cytek) and analyzed with FlowJo 10 software (Tree Star, USA).
7
Cell Surface Staining for Flow Cytometry
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Cells from bone marrow, lung suspension, BALF, and human-induced sputum were prepared as previously described for flow cytometry (Zhang et al. 2021b (link)). For cell surface staining, cells were stained with the antibody for 30 min at 4 °C. A list of the antibodies used in our study is displayed in Additional file 2 : Table S2. Staining quantification was performed using a Cytek Dxp Athena flow cytometer. FlowJo software (version 10, Treestar, Ashland,USA) was used for analysis.
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