96-well plates were seeded with 200 μl of ACE2/HEK293 cells (1.5×104 cells/ml). After a 24 h incubation, recombinant proteins or neutralizing antibody was added. Lentiviral particles capable of transducing 50% of the cells (CoV2-lenti, HKU1-lenti, and 229E-lenti) were added (~25 μl) 30 min later. Following a three-day culture, the ACE2/HEK293 cells were harvested and GFP expression levels determined by BD FACSCelesta™ flow cytometry. Each group contained 2–5 replicates.
Facscelesta flow cytometry
Manufactured by BD
Sourced in United States
The BD FACSCelesta™ is a flow cytometry instrument designed for high-performance cell analysis. It features multi-color detection capabilities and can analyze a wide range of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm kit, by BD (1 mentions)
Dapi solution, by Merck Group (1 mentions)
Prism 9, by GraphPad (1 mentions)
Macsquant analyzer vyb, by Miltenyi Biotec (1 mentions)
Macsquantify software, by Miltenyi Biotec (1 mentions)
Pi cell cycle detection kit, by Keygen Biotech (1 mentions)
Annexin 5 fitc and pi apoptosis detection kit, by Keygen Biotech (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
Dapi ab104139, by Abcam (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Pharm lyse lysing solution, by BD (1 mentions)
Goat anti rabbit igg h l fitc ab6717, by Abcam (1 mentions)
5 protocols using facscelesta flow cytometry
1
Neutralizing Antibody Evaluation for SARS-CoV-2 Entry
2
Multicolor Flow Cytometry of Primary AML Cells
3
Analyzing Multicellular Spheroid Response to Treatments
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Multicellular spheroids were treated with RT, CTx or UniCAR T cell immunotherapy. Forty-eight hours after TM addition, spheroids were washed once with PBS + 2% FBS solution and were trypsinized at 37 °C for 5 min with shaking at 700 rpm. Then, cells were washed with PBS + 0.5% BSA solution, the final volume was adjusted to 100 μL and transferred to a 96-well plate. Six spheroids were used for each measurement. Live and dead cells were distinguished by adding 1 μg/mL of DAPI solution (Sigma-Aldrich, Taufkirchen, Germany). Data was acquired with BD FACS Celesta flow cytometry (BD Bioscience) or MACSQuant VYB Analyzer (Miltenyi Biotec GmbH). All flow cytometric analyses were carried out with FlowJo software (BD Biosciences, Ashland, OR, USA) or MACSQuantify Software (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). The percentage of Cal33 RR viable cells was determined using increasing CD98hc TM concentrations in the coculture assays to calculate the half-maximal effective concentration (EC50) using nonlinear regression fit in GraphPad Prism 9 [32 (link)].
4
Apoptosis and Cell Cycle Analysis
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Cell apoptosis was analyzed by Annexin V-FITC and PI apoptosis-detection kits and Annexin V-PE and 7-amino-actinomycin D (7-AAD) apoptosis-detection kits (KeyGEN Biotech, Nanjing, Jiangsu, China). The cell cycle was analyzed by a PI cell cycle detection kit (KeyGEN Biotech, Nanjing, Jiangsu, China). For the silencing groups, MDA-MB-231 cells stably expressing FOXM1 shRNA and NC shRNA were treated with 4.0 μM Olaparib for 7 days. Cells were stained with Annexin V-PE and 7-AAD according to the manufacturer’s instructions to detect apoptosis. For the drug-treated groups, MDA-MB-231 cells and MDA-MB-468 cells were treated with different concentrations of FDI-6, Olaparib, or their combination for 7 days. After treatment, the cells were collected and stained with Annexin V-FITC and PI following the manufacturer’s protocol to analyze cell apoptosis. Cellular DNA was stained with PI following the manufacturer’s protocol. Cell apoptosis and the cell cycle were detected by BD FACSCelesta flow cytometry (New York, USA). Apoptosis data were analyzed by FlowJo 7.6, and the cell cycle data were analyzed by ModFit LT [31 (link)].
5
Neuronal Cell Isolation and Staining
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Trypsin (27252-018), collagenase II (QN0512), and DMEM (12800-017) were purchased from GIBCO. Fetal bovine serum (04-001-1ACS) was purchased from Biological Industries. Polylysine (P0296) was purchased from Sigma. DAPI (ab104139), anti-NeuN (ab177467), and Goat anti-rabbit IgG H&L (FITC, ab6717) were purchased from Abcam. BD Pharm Lyse lysing solution (No. 555899) was purchased from Becton Dickinson. The stationary liquid 0.01 M PBS solution) contained 3% formaldehyde and 20% sucrose. BD FACS Celesta Flow cytometry was obtained from BD Biosciences-US, and Cytation5 was obtained from BioTek.
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