CAOV-3 cells (5×106) were then incubated with PTX (10 mg/ml), PL-PTX (10 mg/ml) or and or ERK inhibitor (ERKIR; IR7936) and/or anti-TNFα (1:1,000; cat. no. ab6671; 2 mg/ml; both Abcam) treatment for 48 h at 37°C. Following trypsinization, cells were washed in cold PBS and adjusted to 1×106 cells/ml using PBS 3 times at room temperature; CAOV-3 cells were labeled with Annexin V-fluorescein isothiocyanate (V-FITC) and propidium iodide (Annexin V-FITC kit, BD Biosciences) for 2 h at 37°C, according to the manufacturer's protocol, and analyzed with a FACScan flow cytometer (BD Biosciences) using BD FACSChorus™ intuitively designed software version 1.2 (BD Biosciences).
Facschorus
Manufactured by BD
Sourced in United States
The BD FACSChorus is a flow cytometry system designed for research applications. It provides automated sample preparation, analysis, and data management capabilities. The system is capable of analyzing a variety of sample types, including cells, particles, and biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Facsmelody, by BD (5 mentions)
Ab6671, by Abcam (1 mentions)
Annexin 5 fitc kit, by BD (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Pyruvate, by Sartorius (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Cd117 conjugated magnetic beads, by Miltenyi Biotec (1 mentions)
Stepone realtime, by Thermo Fisher Scientific (1 mentions)
Stepone, by Thermo Fisher Scientific (1 mentions)
Moflo astrio, by Beckman Coulter (1 mentions)
Facscalibur, by BD (1 mentions)
Dylight488 maleimide, by Thermo Fisher Scientific (1 mentions)
Antibody labeling kit, by Thermo Fisher Scientific (1 mentions)
Biorad s3, by Bio-Rad (1 mentions)
Melody cell sorter, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
7 protocols using facschorus
1
Apoptosis Induction in CAOV-3 Cells
2
Isolation and Purification of LAG-3+ Cells
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Purified PCs were resuspended at the concentration of 1 × 106 cells per 200 µL PBSX1 and stained with anti-LAG-3 antibody. CD138+ LAG-3pos or LAG-3neg were sorted with a 100-micron nozzle size, and collected into tubes prefilled with RPMI 1640 medium enriched with 20% FCS (10270106, GibcoTM, Thermo Fisher Scientific, Waltham, MA, USA) and 20% Pyruvate (03-042, Biological Industries, Beit-haEmek, Israel). The sorting was done using the BD FACSMelody instrument and BD FACSChorus™ software version 9 (BD Biosciences, Franklin Lakes, NJ, USA). The cells were incubated overnight for recovery.
3
Quantification of Ext1 Gene Expression
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Peritoneal cavity single-cell suspensions were enriched with CD117 conjugated magnetic beads (Miltenyi) and sorted on BD FACSMelody (BD Biosciences) cell sorter running BD FACSChorus (BD Biosciences) software version 1.3.3 after labeling with antibodies described in the flow cytometry section. Sorted cells were incubated with proteinase K (Arrowtec) at 60°C for 5 min to lyse the cells, followed by the extraction of the genomic DNA using the gSYNC extraction kit (Geneaid) following the manufacturer’s instructions. Ext1 and Tfrc genes were amplified together using TaqMan Copy Number Assay ID Mm00613274_cn labeled with FAM and TaqMan Copy Number Reference Assay (cat. # 4458366 labeled with VIC), respectively (both from ThermoFisher) in a PCR thermal cycler (StepOne Real-Time, Applied Biosystems) with the following conditions: 1 cycle of denaturation/enzyme activation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 sec followed by annealing/extension at 60°C for 1 min. Ext1 and Tfrc genes expression were analyzed on StepOne (Applied Biosystems) software version 2.3, and fold increase was calculated using the comparative CT (ΔΔCT) method on Microsoft Excel (Microsoft). Ext1 expression in each sample was normalized using the Tfrc gene expression.
4
Isolation of Vmat2-Cre;tdTomato Cells
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The cell suspensions were analyzed, and 178 Vmat2-Cre;tdTomato cells were sorted onto a 384 Smart-Seq2 well plate using a BD FACSMelody and BD FACSChorus software. The particles were initially gated based on the area in forward and side light scatter (FSC-A/SSC-A) to detect cell-sized particles of homogenous character, which were further gated on height and width in two steps (1. SSC-H/SSC-W, 2. FSC-H/FSC-W) to isolate single-cells. To detect tdTomato positive cells and exclude single-cell like particles with autofluorescence, the single-cells were gated on the intensity of different settings of the red and far red channels (PE-Cy5(YG)-A, PE-Cy7(YG)-A, (YG)-A) (Figure S6 ). The cortical sample from the tdTomato negative animal was used as a control sample to detect baseline intensity of the red and far red channels (Figure S6 A), where after the tdTomato intensity threshold could be detected in the (YG)-A channel (Figure S6 B). The tdTomato positive single-cells were thereafter sorted onto the plate, which was stored at − 80 °C until library sequencing.
5
Flow Cytometry Analysis of Lung Cells
6
Quantitative Analysis of Cell Surface Binding
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For binding studies PMT was labelled covalently with DyLight488 maleimide (Thermo Fisher Scientific) according to the manufacturer‘s instructions. Pre-cultured HeLa, HepG2 or MEF cells and derivates were detached from culture plates using 10 mM EDTA in PBS. For each condition 2 × 105–2.5 × 105 cells were used and incubated for 20 min with the indicated PMT-DyLight488 concentrations at 4°C. Following several washing steps with ice-cold TBS, cell bound fluorescence was measured using a FACS Melody (BD Bioscience). Via the appropriate Software FACSChorus (BD Bioscience) 10.000 cells were analyzed. After gating and the average of surface bound fluorescence of all cells was given.
For analysis of surface bound fluorescence of cells, the median forward scatter of the analyzed Hela and HepG2 cell populations was measured for normalization.
For analysis of surface bound fluorescence of cells, the median forward scatter of the analyzed Hela and HepG2 cell populations was measured for normalization.
7
Immune Cell Identification and Sorting
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Single cells suspensions were incubated in PBS containing 0.5% FCS and 10 mM EDTA with the indicated antibodies (Supplementary Table 1 ) for 20 min at 4 °C. For the identification of tetramer-binding CD8+ T cells, cells were stained with PE-labeled H-2Kb-TRP2180–188 tetramer (MBL, Woburn, MA) for 10 min at 37 °C. For discrimination between live and dead cells, single-cell suspensions were stained with AQUA-BV510 or e780 in PBS prior to antibody staining (Molecular Probes) or by adding 7AAD prior to acquisition (Calbiochem). For intracellular or intranuclear staining, cells were fixed and permeabilized using the BD Cytofix/Cytoperm (BD Biosciences) or the FoxP3 Transcription Factor (eBiosciences) kits, respectively. The anti-LCMV-NP antibody was conjugated with AF657 or AF594 (Antibody Labeling Kit, ThermoFisher). Cells were acquired with a BD LSRFortessa (BD Biosciences) and data analysis was performed using FACS Diva (BD Bioscience, v8.0.1) and FlowJo (Treestar Inc., v10). Cells were sorted on a Biorad S3 (Biorad) or BD Melody Cell Sorter and data were analyzed using FACSChorus (BD Biosciences, v1.3).
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