PBS-washed EBs were incubated (37°C, 3–5 min) in TrypLE Express (Gibco). PBS + 10% FBS + 1% P/S was added and single cells suspended (P1000 pipette). 106 cells per 100 μL were immunostained (30 min on ice) (Table S1 ). Dead cells were Hoechst 33342 (Invitrogen) excluded. LSRFortessa, FACScan, FACSAria III/Fusion (Becton Dickinson) and FlowJo software (TreeStar) were used. Strategy for detection of GFP and/or Venus is described in Supplemental Experimental Procedures and Figure S6 .
Facsaria 3 fusion
Manufactured by BD
The BD FACSAria III FUSION is a high-performance cell sorter. It is designed for multi-parameter flow cytometry analysis and sorting of cells. The instrument features advanced optics, fluidics, and electronics to enable precise cell sorting and data acquisition.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Facscan, by BD (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Pneumacult ali s medium, by STEMCELL (1 mentions)
Collagen 4, by Merck Group (1 mentions)
Pneumacult ex plus medium, by STEMCELL (1 mentions)
Collagen 1, by Corning (1 mentions)
Kaluza analysis software version 2, by Beckman Coulter (1 mentions)
Recombinant mouse ifn γ, by BioLegend (1 mentions)
Compbead, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Anti ki67, by BD (1 mentions)
Lsrfortessa x 20 flow cytometer, by BD (1 mentions)
Hoechst 33342 ready flow reagent, by Thermo Fisher Scientific (1 mentions)
Ls magnetic column, by Miltenyi Biotec (1 mentions)
Facsaria 2, by BD (1 mentions)
7 protocols using facsaria 3 fusion
1
Flow Cytometry Analysis of Engineered Cells
2
Expansion and Differentiation of Human Airway Epithelial Cells
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Human ABCs were cultured in PneumaCult™-Ex Plus Medium (STEMCELL Technologies) in T25 flasks, pre-coated with collagen I (Corning), at 37 °C, 95% O2, and 5% CO2. After two weeks of culturing, cells were seeded onto Corning® Transwell® Cell Culture Plate (Corning), pre-coated with collagen IV (Sigma-Aldrich), at a density of 30,000 cells per well. In the first, expansion phase, 100 µl of PneumaCult™-Ex Plus Medium (STEMCELL Technologies) were added to the cells and 600 µl were added to the bottom chamber. After reaching confluency (7–10 days), the medium was aspirated from the upper chamber and the medium from the bottom chamber was replaced by PneumaCult™-ALI-S Medium (STEMCELL Technologies), to promote the second, differentiation phase, characterized by maturation of pseudostratified mucociliary epithelium. Medium change was performed every other day. Experiments were performed with ALI-cultures after ciliar beating was visually detectable (~4 weeks after ALI). Cells were then harvested and stained for EPCAM and PROM1. PROM1+ cells were sorted by flow cytometry (Becton-Dickinson FACSAria III Fusion).
3
Membrane Labeling and Cell Sorting
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For membrane labeling, TILs and cancer cells were stained with fluorochrome-coupled mAbs incubated for 20 minutes at 4°C, protected from light, and then washed with 1× PBS. Intracellular staining was performed after permeabilization with the FoxP3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, catalog 00-5523-00) and intracellularly labeled with anti-Foxp3 mAb (eBiosciences, clone PCH101) and anti-Ki67 (BD Biosciences, catalog 556027), following the manufacturer’s protocol. Cell samples were acquired on a BD LSRFortessa X-20 flow cytometer (BD Biosciences) with single-stained Ab-capturing beads used for compensation (CompBeads, BD Biosciences, catalog 552843). For cell sorting, Zombie Aqua– cells and anti-HLA-I+ and HLA-I– cells after BCG coincubation for 24 hours were sorted on a BD FACSAria III Fusion (BD Biosciences). Data were analyzed with Kaluza Analysis software, version 2.1 (Beckman Coulter). MB49 and UPPL1541 cell lines were cultured in vitro into media with or without recombinant mouse IFN-γ (BioLegend, catalog 575306) for 24 hours and subsequently stained for HLA-I with FITC anti-mouse H-2Kb Ab (BioLegend, catalog 1165005) and run by flow cytometry.
4
Characterizing EGF-Receptor Expression
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Cells were prepared by adding EGF-free media for 30 min before lifting and suspending cells in 0.1% BSA/PBS. Cells were incubated in 100 ng/ml EGF-647 (E35351, Thermo Fisher Scientific) in 0.1%BSA/PBS, with cells incubated in 0.1% BSA/PBS as a negative control, for 25 min. Cells were washed three times in 0.1%BSA/PBS before being analysed on the BD FACSAria III FUSION. Where indicated, cells were sorted by EGF-647 gated into high and low groups, and a sort check was performed to verify these were true populations prior to expanding these cells onto 22×22 mm2 cover-slips. Fifteen days after the cells were sorted, the slides were fixed, permeabilized, and DNA FISH performed as above.
5
Quantitative EGF Receptor Profiling
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Cells were prepared by adding EGF-free media for 30 minutes before lifting and suspending cells in 0.1% BSA/PBS. Cells were incubated in 100ng/ml EGF-647 (E35351, Thermo Fisher Scientific) in 0.1%BSA/PBS, with cells incubated in 0.1% BSA/PBS as a negative control, for 25 minutes. Cells were washed three times in 0.1%BSA/PBS before being analyzed on the BD FACSAria III FUSION. Where indicated, cells were sorted by EGF-647 gated into high and low groups, and a sort check was performed to verify these were true populations prior to expanding these cells onto 22x22mm cover slips. Fifteen days after the cells were sorted, the slides were fixed, permeabilized and DNA FISH performed as above.
6
Hoechst 33342 Cell Cycle Analysis
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Two drops of Hoechst 33342 Ready Flow Reagent from Invitrogen was added to 2 × 106 cells and incubated for 15 min. Cells were spun, resuspended in Miltenyi Biotec FACs buffer and assayed using the BD FACSAria Fusion 3. Green fluorophores were excited at 488 nM with emission at 530 nM, whereas orange fluorophores were excited at 561 nM with emission at 586 nM. Fluorophores in Phenomex Lightning were detected using the FITC and TRED excitation/emission filters in the Cell Analysis Suite. Hoechst staining was measured at 375 nM with emission at 450 nM. To determine FACs signal overlap between fluorophores and Hoechst, 10,000 cell measurements were read into R, Hoechst signal was split into 100 bins and cell cycle fluorophores scaled from 1 to 100 and intensities were compared.
7
Isolation and Purification of KL Cells
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Bone marrow was harvested from male transgenic mice 20 weeks post tamoxifen administration. Bone marrow cells were incubated with ACK red cell lysis buffer to remove erythrocytes and depletion of lineage positive cells was performed using the MACS Lineage Cell Depletion kit for mouse (Miltenyi Biotech, Cat#130–090–858) and magnetic LS columns (Miltenyi Biotech, Cat#130–042–401) according to the manufacturer’s instructions. Lineage depleted cells were stained with fluorophore-conjugated antibodies targeted against cell surface markers (Supplementary Table 1 ) as above and 4′,6-Diamidino-2-Phenylindole (DAPI) was used as a viability marker. Viable KL (cKit+Sca1−Lin−) were sorted on BD FACSAria Fusion 5, BD FACSAria II and BD FACSAria Fusion 3 (BD Biosciences) and used for transplantation and molecular analyses.
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