Tumor cells from monolayers or tumorsphere cultures (1 × 106) were incubated for 1 h in the presence or absence of 10 µM Hoechst 33342 (Sigma‐Aldrich). In some cases, to block Hoechst efflux, cells were stained in the presence of 50 μM verapamil (Sigma‐Aldrich), as previously described.29 The cells were then washed twice with PBS, and the percentage of cells that excluded the dye was analyzed using a FACSAria IIu flow cytometer (BD Biosciences). The data obtained were analyzed using FlowJo 10 software.
Facsaria iiu flow cytometer
Manufactured by BD
Sourced in United States
The BD FACSAria IIu is a high-performance flow cytometer designed for advanced cell analysis and sorting. It is capable of multiparameter detection and analysis of cells and particles in suspension.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 apc h7, by BD (3 mentions)
Anti hla dr pe, by BD (3 mentions)
Anti cd4 pecf594, by BD (3 mentions)
Anti cd8 apc, by BD (3 mentions)
Facsdiva software, by BD (2 mentions)
Anti cd38 bb515, by BD (2 mentions)
Trustain fcx affinity purified anti mouse cd16 32 fc block, by BioLegend (2 mentions)
Facs caliber cytometer, by BD (2 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Verapamil, by Merck Group (1 mentions)
Cellquest software, by BD (1 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Mouse anti flag m2 antibody, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
D pbs, by American Type Culture Collection (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Alexa 647 conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
22 protocols using facsaria iiu flow cytometer
1
Hoechst 33342 Efflux Analysis
2
Cell Cycle Analysis of RP4010 Treatment
3
Quantifying FLAG-tagged Receptor Expression
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Recombinant HEK cells expressing the FLAG-tagged human ETA variants were seeded at 106 cells per well (in presence or absence of 100 ng/ml tetracycline) into 6-well plates in growth medium and incubated overnight at 37°C in 5% CO2. Then, the cells were washed with PBS and detached with cell dissociation buffer (Life Technologies, USA). FLAG-hETA receptors were stained with mouse anti-FLAG M2 antibodies (Sigma, USA), diluted in PBS/2 mM EDTA/0.5% fatty acid free BSA (Calbiochem, Germany). As secondary antibody Alexa Fluor 488 goat anti-mouse IgG (Life Technologies, USA) was used. Then, mean fluorescence intensities (MFI) of the propidium iodide-negative cells were recorded using a FACSAria IIu flow cytometer (BD Biosciences, USA). Surface receptor expression was calculated by subtracting the MFI of the non-induced cell pools from the MFI of the tetracycline-induced cells pools and then comparing this value to the value obtained for the wildtype-expressing cells (100%).
4
Evaluating TLR2 Expression in HEK-Blue Cells
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The unstimulated, stimulated, neutralized, and immunomodulated HEK-BlueTM cell cultures were trypsinized (trypsin-EDTA, Gibco, Gaithersburg, MD, USA) and removed from the 24-well plates in which they were seeded. After 5 min centrifugation at 200× g, the cell pellet was resuspended in D-PBS (ATCC, Virginia, USA) and incubated for 20 min with FITC anti-TLR2-conjugated monoclonal antibodies (5 µL, Immunostep, Salamanca, Spain). TLR2 expression was evaluated using FACSAria iiu flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with FACSDiva software (BD Biosciences, Franklin Lakes, NJ, USA). To avoid that the background from the antibody may interfere with the study, samples were washed with D-PBS and centrifuged before being analyzed. Unlabeled and thus non-fluorescent cells, as well as HEK-BlueTM Null1 cells, were used as both negative controls. TLR2 expression was reported as AFU.
5
Monocyte and Neutrophil TLR2/4 Expression
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TLR2 and 4 expression analyses were performed by flow cytometry in blood samples, withdrawn from all KC patients and control subjects, collected in EDTA-anticoagulated tubes. For the expression analysis of TLR2 and TLR4, monocytes, lymphocytes and neutrophils were separated by their forward and side scattering signal characteristics. Fluorescein isothiocyanate (FITC) anti-TLR2-conjugated monoclonal antibodies (IMMUNOSTEP, Salamanca, Spain) and phycoerythrin (PE) anti-TLR4 conjugated monoclonal antibodies (IMMUNOSTEP, Salamanca, Spain) were used to quantify TLR expression. Samples were analyzed on a FACSAria iiu flow cytometer (BD Biosciences, NJ, USA). Cell fluorescence was measured immediately after staining, and data were analyzed with the use of FACSDiva software (BD Biosciences, NJ, USA). Mean expression of TLR2 and TLR4 in monocytes and neutrophils was expressed as AFU (arbitrary fluorescence units).
6
ALDH1+ Cell Population Isolation
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The ALDEFLUOR kit (Stem Cell Technologies, 1700) was used to identify and sort cell populations with a high ALDH1 enzymatic activity per the manufacturer's instructions. In brief, cells were treated with or without EPZ-5676 for the indicated time and harvested with TrypLE. Cells (1 × 106) were suspended in ALDEFLUOR assay buffer with ALDH substrate (BODIPY-aminoacetaldehyde-diethyl acetate or BAAA, 1 mmol/L). The ALDH inhibitor diethylaminobenzaldehyde (DEAB) was used to control background fluorescence as negative control. The samples were incubated for 45 minutes at 37°C. ALDEFLUOR was excited at 488 nm, and fluorescence emission was detected using standard fluorescein isothiocyanate (FITC) 530/30-nm band-pass filter using the CantoII flow cytometer (BD Biosciences). To sort ALDH1+ and ALDH1− cell populations, 10–20 × 106 MDA-MB-468 cells were subjected to the ALDEFLUOR assay as above. Cells were aliquoted as 1 × 106 cells/tube in 1 mL of ALDEFLUOR assay buffer. After incubation, samples were resuspended in assay buffer/tube on ice and then filtered through the cell strainer cap into 5 mL polystyrene flow cytometry tubes. Samples were then sorted using FACSAria IIu flow cytometer (BD Biosciences) and collected in ice-cold PBS in 5 mL polypropylene tubes, centrifuged, and plated into overnight culture to facilitate removal of dead cells and debris.
7
Flow Cytometry-Based Viability and GFP Knockdown
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For cell viability evaluation, samples were analyzed on an Accuri C6 Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). For eGFP knockdown analysis, a FACSAria IIu Flow Cytometer (Becton Dickinson) was used, operating under BD FACSDiva Software Version 6. Cells were illuminated with a 488-nm laser line, and the fluorescence was determined using a 530 ± 30 emission filter. The viable population was gated by side scatter/forward scatter evaluation, and the mean fluorescence intensity of duplicate samples was calculated. Mean values of chol-siGFP-treated cells were corrected for background fluorescence by subtracting wild-type cell measurements, and for non-specific treatment effects by normalizing to samples treated with control chol-siRNA but otherwise identical conditions. The gating strategies used are shown in Supplementary Fig. 8 .
8
Flow Cytometry Analysis of Histone Modifications
9
Characterization of Epithelial Cell Phenotypes
10
Quantifying CD38 and HLA-DR on CD8+ T cells
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To quantify the expression of CD38 and HLA-DR on CD8+ T cells, cryopreserved PBMC were thawed and stained with the following antibodies: anti-CD3 APC-H7, anti-CD4 PECF594, anti-CD8 APC, anti-CD38 BB515, and anti-HLA-DR PE (BD Biosciences, United States), and acquired using a BD FACSAria IIu Flow Cytometer (BD Biosciences, United States). The Fixable Viability Stain 450 (FVS 450-BD Biosciences, United States) was used to exclude non-viable cells. Flow cytometric analyses were performed with FlowJo v.10.0.7 (Tree Star Inc., Ashland, OR, United States). Plasmatic levels (pg/mL) of IP-10, IL-18, RANTES, CTACK, and PDGF-AA were measured using the human Magnetic Luminex Performance Assay (R&D systems, United States), following manufacturer’s instruction and the analyses were performed on a Luminex 200 System (Luminex, United States).
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