H1299 and PC9 cells (1 × 105) were cultured in six‐well plates and transfected with siFOXM1 or NC siRNA, or treated with 5 µM TST or DMSO. The fluorescein isothiocyanate Annexin V Apoptosis Detection Kit I was used to stain the cells (#556547; BD Biosciences). The cells were analyzed within 1 h to reduce any adverse effects from staining. Flow cytometry was performed using a FACSVerse Cell Analyzer (BD Biosciences).
Facsverse cell analyzer
Manufactured by BD
Sourced in United States
The BD FACSVerse Cell Analyzer is a flow cytometry instrument designed for multiparameter analysis of cells. It utilizes laser excitation and fluorescence detection to provide high-resolution data on various cellular parameters, including size, granularity, and fluorescent labeling.
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Lab products found in correlation
Facsuite software, by BD (2 mentions)
Apc conjugated anti mouse igg antibodies, by Jackson ImmunoResearch (2 mentions)
Annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Anti f4 80 apc, by BioLegend (1 mentions)
Anti mhc 2 fitc, by BioLegend (1 mentions)
Cell discoverer microscope, by Zeiss (1 mentions)
Alexa fluor 647 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Histofix, by Carl Roth (1 mentions)
Facs buffer, by BD (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Btx ecm 830 system, by Harvard Apparatus (1 mentions)
Apoptosis detection kit, by Dojindo Laboratories (1 mentions)
Aldefluor, by STEMCELL (1 mentions)
Annexin 5, by BioLegend (1 mentions)
Aldefluor, by BD (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Cd3 pe, by BioLegend (1 mentions)
Cd45r b220 pe cy7, by BioLegend (1 mentions)
Cd49d apc, by BioLegend (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
17 protocols using facsverse cell analyzer
1
Apoptosis Analysis of FOXM1 Silencing
2
Quantifying Macrophage Polarization by Flow Cytometry
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RAW264.7 cultured cells were harvested by centrifugation at 3000 rpm at 4°C for 5 minutes and subsequently labeled with anti-NOS2-PE (696805, Biolegend, USA), anti-MHC-II-FITC (107606, Biolegend, USA), and anti-F4/80-APC (123116, Biolegend, USA). Flow cytometry was performed with a FACS Verse™ Cell Analyzer (BD Biosciences, USA), and the data were analyzed in FlowJo software (Tree Star, USA) to determine the percentage macrophage polarization (iNOS+F4/80+ or MHC-II+F4/80+).
3
Binding of AAT to Candida albicans
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C. albicans was seeded in 8-well ibidi µ-slides at 3 × 104 CFU/well and grown for 2 h in RPMI. Afterwards, AAT was added to a concentration of 1 mg/mL for another hour under the same conditions. Cells were carefully washed with PBS and fixed 20 min in 4% Histofix (Roth). Cells were washed and blocked in PBS containing 1% BSA for 30 min RT. The primary antibody (unlabelled polyclonal rabbit IgG anti-AAT #711079, Invitrogen) was incubated diluted 1:200 overnight at 4 °C. Cells were washed with PBS, stained for 2 h at RT with 20 µg/mL goat-anti-rabbit-AlexaFluor647 (Invitrogen) and washed again. After the last washing step, the fixed C. albicans cells were covered with PBS and imaged on a Cell discoverer microscope (Zeiss) at 40× magnification.
For flow cytometry detection, SC5314 yeasts were cultured shaking at 37 °C in presence of 1 mg/mL AAT for 1 h. Cells were washed with FACS buffer (PBS, 2% FCS) and stained with anti-AAT (unlabelled polyclonal rabbit IgG, Invitrogen) at 4 °C overnight. The next day, cells were washed with FACS buffer and stained with goat-anti-rabbit-AlexaFluor647 (Invitrogen) at 4°C for 20 min. Cells were filtered through a 70 µm mesh before acquisition on a FACSVerse Cell Analyzer (BD Biosciences, Franklin Lakes, NJ). Analysis was performed using FlowJo V10.
For flow cytometry detection, SC5314 yeasts were cultured shaking at 37 °C in presence of 1 mg/mL AAT for 1 h. Cells were washed with FACS buffer (PBS, 2% FCS) and stained with anti-AAT (unlabelled polyclonal rabbit IgG, Invitrogen) at 4 °C overnight. The next day, cells were washed with FACS buffer and stained with goat-anti-rabbit-AlexaFluor647 (Invitrogen) at 4°C for 20 min. Cells were filtered through a 70 µm mesh before acquisition on a FACSVerse Cell Analyzer (BD Biosciences, Franklin Lakes, NJ). Analysis was performed using FlowJo V10.
4
Quantification of Leishmania Parasites by Flow Cytometry
5
Isolation and Characterization of Adipose Tissue Macrophages
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The macrophages were isolated from the white adipocyte tissue according to a previous study (20 (link)). The white adipocyte tissue was excised immediately after sacrifice and the fat tissue was minced, using a razor blade, into small pieces for collagenase-buffer–dependent digestion (2.5% HEPES, 10 mg/mL bovine serum albumin, 3 mg/mL (0.3%) collagenase type II in Dulbecco's modified Eagle medium (DMEM) with 4.5 g/L glucose without L-glutamine and sodium pyruvate). The minced-tissue and digestion-buffer mixture was incubated under slow continuous rotation at 37 °C for 45 min, and the digested tissue was then filtered through a 100 μm cell strainer into a collection tube. The cells were washed twice with cold DMEM and, if necessary, erythrocytes were lysed using red blood cell (RBC) lysis buffer. The isolated cells were resuspended in cold phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS), and 1 × 106 cells were transferred for flow cytometry (BD FACSVerse Cell Analyzer, BD bioscience, CA, USA) after staining with anti-mouse CD11b, F4/80 or CD206 antibodies for 40 min at 4 °C, to identify macrophages.
6
In-cell NMR Measurement of Oligonucleotide Uptake
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A sample for in-cell NMR measurement was prepared as described in (16 (link)). Briefly, HeLa cells were electroporated with 400 μM non-labelled DNA oligonucleotide (d-AAGGGTGGGTCCCACCCTGGGTGGGT) supplemented with 10 μM 5′-FAM labelled oligonucleotide in electroporation buffer (140 mM Na2HPO4/NaH2PO4, 5 mM KCl, 10 mM MgCl2, pH 7.0), using electric pulses 1000 V, 100 μs followed by 350 V, 30 ms (BTX-ECM 830 system; Harvard Apparatus, USA). After electroporation, FAM intensity in cells was monitored with flow cytometry (BD FACS Verse Cell Analyzer; BD Biosciences, USA) to check transfection efficiency and propidium iodide staining was used to determine cell survival. Subcellular localization of the introduced oligonucleotide and cell morphology was monitored using confocal microscopy (LSM 800; Carl Zeiss, Germany). For in-cell NMR, the cells were suspended in Leibovitz L15–/– medium containing 10% D2O, transferred into a 5 mm NMR cuvette, and pelleted by manual centrifugation to concentrate in active coil volume. 1D 1H in-cell NMR spectra were acquired at Bruker Avance NEO 950 MHz NMR (Bruker Corporation, Billerica, MA, USA) using a 1D 1H JR-echo (1–1 echo) pulse sequence (17 ) with zero excitation set to the resonance of water and the excitation maximum set to 12.5 ppm.
7
Immunophenotyping and Apoptosis Assay
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After cell counting, cells were washed with PBS and stained with antibodies from BD Biosciences (Franklin Lakes) against CD3-FITC (555916), CD4-PE (561843), and CD8-PE (561949). For the cell apoptosis assay, cells were stained with the Apoptosis Detection Kit (AD10; Dojindo) according to the manufacturer’s instructions, and annexin V was measured by fluorescent signal FITC. The cells were then washed again with PBS and resuspended with 2% FBS in PBS. The cell samples were injected into the BD FACSVerse Cell Analyzer (BD Biosciences), and the flow cytometry standard data were analyzed using FlowJo software (version 10.0.7; BD Biosciences).
8
Apoptosis Analysis of Cells Treated with RFP and H2O2
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Cells (10 × 10⁴ cells/well) were seeded in 6-well plates for 24 h, then treated with 2,500 μg/ml of RFP for another 24 h. H2O2 was administrated at proper concentration for 18 h. Cells were collected and washed three times with pre-cold phosphate-buffered saline. Then cells were re-suspended in the binding buffer containing annexin V-FITC and propidiumiodide (PI). After incubation for 20 min at room temperature, BD Facsverse Cell Analyzer (BD Biosciences, San Jose, CA, United States) was used to perform flow cytometry analyses.
9
ALDH Activity and CD133 Expression
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ALDH enzymatic activity was assessed as previously described [34 (link)–36 ], using ALDEFLUOR (Stem Cell Technologies, Seattle, WA) according to the manufacturer’s instructions. Following ALDH staining, cells were incubated with CD133-APC antibody (BD Biosciences, Ashland, OR) at 1:20 dilution in ALDEFLUOR buffer for 25 min on ice, protected from light. Cells were washed in PBS and resuspended in 400 µl PBS for analysis on a BD FACSVerse cell analyzer (BD Biosciences, Franklin Lakes, NJ). Cell death was assessed by Annexin V (640,905, Biolegend, San Diego, CA) and propidium iodide (PI) (R37169, Thermo Scientific, Waltham, MA) staining on cells treated as indicated, as previously described [35 , 36 ].
10
Delivery Efficiency of ASO or Toc-HDO in T Cells
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The delivery efficiency of ASO or Toc-HDO into primary T cells in vivo and EL4 in vitro cells was assayed by flow cytometry using Alexa Flour 647-labeled ASO. Cells were harvested at the indicated time points and washed in PBS by centrifugation at 500 ×;g for 5 min. The cells were then washed and suspended in PBS with 2% BSA and 0.05% sodium azide (Sigma-Aldrich). Surface marker expression levels were determined using the following antibodies: CD3-PE (BioLegend, clone 17A2, 100205), CD45R/B220-PECy7 (BioLegend, clone RA3-6B2, #103221) or CD45R/B220-FITC (BioLegend, clone RA3-6B2, #103205), and CD49d-APC (BioLegend, clone R1-2, #103621). All related antibodies are listed in Supplementary Table 2 . Cells were then sorted using a BD FACSAria cell sorter, BD FACSDiva software, and FlowJo v10 software (BD Biosciences) or analyzed using a BD FACSVerse cell analyzer and BD FACSuite software (BD Biosciences). The sorting/gating strategy is shown in Supplementary Fig. 2g .
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