BE(2)-C cells were seeded in 3D scaffolds 24 h prior to treatment. Spheroids were treated for 48 h with solvent control (DMSO), doxorubicin (500 ng/ml), combinations of doxorubicin with bufexamac (30 μM), chloroquine (5 μM) or both. For the knockdown approach, BE(2)-C cells were seeded in 3D scaffolds three days after transfection with ATG5, FOXO3a, HDAC6, HDAC10 or control siRNAs, grown for an additional 48 h and treated with 500 ng/ml doxorubicin 24 h before staining. For FACS analysis, chips were washed with RPMI w/o phenol red (10% FCS), transferred to a fresh 6-well plate, trypsinized (4 min, 37 °C), centrifuged and washed again in cold medium w/o phenol-red (10% FCS). For the analysis of 2D conditions, BE(2)-C cells were seeded in monolayers in six-well plates 24 h prior to treatment. Thereafter, the same protocol as for the 3D scaffolds was followed. Doxorubicin fluorescence was quantified on a BD FACSCanto II platform using a Phycoerythrin (PE) filter setting.
Facscanto 2 platform
Manufactured by BD
Sourced in United States
The BD FACSCanto II platform is a flow cytometer designed for multicolor analysis of cells and particles. It has the capability to simultaneously detect and analyze multiple fluorescent parameters on individual cells or particles in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Cyto id, by Enzo Life Sciences (1 mentions)
Alexa 488 labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ebiosciencetm foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Vicell xr automatic cell counter, by Beckman Coulter (1 mentions)
Annexin 5 propidium iodide apoptosis kit, by MultiSciences Biotech (1 mentions)
Rnase a, by Merck Group (1 mentions)
Facsdiva 7, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
Annexin 5 pi apoptosis detection kit, by Dojindo Laboratories (1 mentions)
Pe conjugated anti human igg fc, by BioLegend (1 mentions)
Facsaria 3, by BD (1 mentions)
Fitc annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Camptothecin, by Merck Group (1 mentions)
Dq ovalbumin, by Thermo Fisher Scientific (1 mentions)
Fix perm cell permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Live dead cell viability assay, by Thermo Fisher Scientific (1 mentions)
Cd16 apc cy7, by BioLegend (1 mentions)
Apc hla g, by BioLegend (1 mentions)
Alexa fluor 488 antibody labeling kit, by Thermo Fisher Scientific (1 mentions)
18 protocols using facscanto 2 platform
1
Evaluating Doxorubicin Combination Treatments in 3D Neuroblastoma Model
2
Lysosomal Quantification in SK-N-BE(2)-C Cells
3
Quantifying Autophagy in 3D Cell Cultures
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Three days after siRNA transfection, BE(2)-C cells were seeded in 3D scaffolds and grown for an additional 72 h. Where indicated, cells were treated with 5 μM CQ the night before staining with CYTO-ID (Enzo Life Sciences). For staining (6 d post-siRNA transfection), chips were washed once with medium w/o phenol red, transferred to a fresh 6-well plate and covered with 500 μl CYTO-ID staining solution (1 × assay buffer, 5% FCS, 1 μl CYTO-ID) for 30 min at 37 °C. Cells were washed with medium w/o phenol red, trypsinized (4 min. 37 °C), centrifuged and washed again in cold medium w/o phenol-red. CYTO-ID fluorescence was quantified on a BD FACSCanto II platform using an Alexa488 filter setting
4
Quantifying Lysosomal Activity via Flow Cytometry
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Six days after siRNA transfection or 24 h after treatment with HDAC or lysosomal inhibitors, cells were stained for 1 h with 50 nM LysoTracker® Red DND-99 in medium under standard cell culture conditions. Cells were washed with ice-cold RPMI w/o phenol-red and detached using Trypsin/EDTA for 3 minutes at 37 °C. Cells were centrifuged, resuspended in RPMI w/o phenol red and LysoTracker® Red fluorescence was quantified on a BD FACSCanto II platform using the PE filter setting.
5
Quantification of P-gp, LAMP-1, and DNA Damage
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Cells were dissociated from dishes using non-enzymatic cell dissociation reagent for five minutes. Cells were counted using a ViCell XR automatic cell counter (Beckmann Coulter) and 2.5 ∗ 105 cells were transferred into a pre-cooled 96-well round bottom plate (TPP92097). Cells were stained for 1.5–2 hours on ice with primary anti P-gp (Biozol ABA-AB00143-1.1), LAMP-1 (H4A3, DHSB) antibody or isotype control (DLN-05794, Dianova, Barcelona, Spain) and for 1 hour with APC-labeled secondary antibody (Dianova 115-136-068). Cells were washed three times in FACS buffer (5% FBS in PBS) after each antibody incubation step. For evaluation of DNA double-strand breaks, cells were detached from dishes using trypsin, counted using a ViCell XR automatic cell counter (Beckmann Coulter) and transferred into a pre-cooled 96-well round bottom plate (4 ∗ 106 cells/well). Cells were fixed and permeabilized using the eBioscienceTM Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific). Cells were stained for 1.5 h with Phospho-Histone H2A.X (S139) primary antibody (CST #9718) and for 1 h in Alexa-488-labeled secondary antibody (ThermoFisher A-11008). Cells were washed three times in 1× permeabilization buffer after each antibody incubation step and fluorescence was quantified on a BD FACSCanto II platform.
6
Annexin V/PI Apoptosis Assay for SKM-1 Cells
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The apoptosis rate of treated skm-1 cells was evaluated using an annexin
V/propidium iodide (PI) apoptosis kit from MultiSciences Biotech (Zhejiang,
China) according to the manufacturer’s protocol. For each drug concentration, 1
to 3 × 106 cells were used on the BD FACSCanto II platform (BD
Biosciences, San Jose, CA, USA). Intact cells were marked as annexin
V–/PI–, whereas early apoptotic cells and late
apoptotic/necrotic cells were marked as annexin V+/PI– and
annexin V+/PI+, respectively.
V/propidium iodide (PI) apoptosis kit from MultiSciences Biotech (Zhejiang,
China) according to the manufacturer’s protocol. For each drug concentration, 1
to 3 × 106 cells were used on the BD FACSCanto II platform (BD
Biosciences, San Jose, CA, USA). Intact cells were marked as annexin
V–/PI–, whereas early apoptotic cells and late
apoptotic/necrotic cells were marked as annexin V+/PI– and
annexin V+/PI+, respectively.
7
Cell Cycle Analysis by Flow Cytometry
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Cells were fixed in ice-cold 70% ethanol for 1 h, washed with 38 mM Na-citrate buffer and stained with 50 µg/mL propidium iodide (PI) containing 50 µg/mL RNAse A (Sigma-Aldrich) at 37 °C for 20 min. Measurement of DNA content was conducted using BD FACSCanto II platform (BD Bioscience, Franklin Lakes, NJ, USA), the data was analyzed using FlowJo™ v10.6.1 software (BD Bioscience).
8
Evaluating PHF6 Effect on T-cell Differentiation
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The effect of PHF6 on the CD4/CD8 T-cell differentiation was evaluated using flow cytometry. The monoclonal antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA) and comprised CD1a APC (559775), CD2 PE-Y7 (335821), CD3 V-450 (558117), CD4-PECP 515 (332772), CD7 PE (332774), and CD8 APC-H7 (641400), and the TdT-FITC (F7139) was from Dako Denmark (Glostrup, Hovedstaden, Denmark). The DND41 cells were analyzed for T-cell surface antigens CD1a, CD2, CD3, CD4, CD7, CD8, and TdT in accordance with the current guidelines for the classification of leukemia by flow cytometry [22 (link),23 (link)]. Next, 2 × 104 cells per 1 μL were counted for analysis. One group of cells was transfected with the wild-type PHF6 expression plasmid with lipofectamine, while the control group was not transfected. After 24 h of incubation at 37 °C, the cells were centrifuged at room temperature, and the cell pellets were resuspended in 1 mL of FBS and 4 mL of 1 × PBS. The cells were then labeled with anti-CD2, CD1a, CD3, CD4, CD7, CD8, and TdT antibodies. Flow cytometric analysis was performed according to the manufacturer’s instructions using BD FACSDiva 7.0 software (Becton Dickinson, Mississauga, ON, Canada) in the BD Facs Canto II platform (Indianapolis, IN, USA).
9
Retrospective Analysis of HSTCL
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A retrospective analysis of all the consecutive patients of HSTCL diagnosed through flow cytometry over a 6year period (July 2013 to June 2019) was carried out. Relevant clinical history along with hematological and biochemical profiles of all the patients was retrieved from hospital electronic medical records.
For morphological evaluation, PB smears, BM aspirates, and biopsies stained with Leishman, May-Grunwald-Giemsa, and hematoxylin and eosin stains, respectively, were reviewed. FCI using 4-8 color panels were performed on PB or BMA samples. A stain-lyse-wash protocol was used with following monoclonal antibodies: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD23, CD34, CD38, CD45, CD56, CD200, FMC7, αβ TCR, γδ TCR, kappa light chains, and lambda light chains in different tube combinations. Briefly, 100-300 μl of BMA/PB samples was incubated with antibody cocktails for 20-30 min in dark. It was then washed with phosphate-buffered saline to remove the excess of unbound antibodies. This was followed by red cell lysis using commercial lysing agents, followed by wash and a final suspension of cell pellet in paraformaldehyde. The processed samples were acquired on a BD FACS Canto II platform with FACS Diva software (BD Biosciences, San Jose, CA, USA), which was also used for the analysis of samples.
For morphological evaluation, PB smears, BM aspirates, and biopsies stained with Leishman, May-Grunwald-Giemsa, and hematoxylin and eosin stains, respectively, were reviewed. FCI using 4-8 color panels were performed on PB or BMA samples. A stain-lyse-wash protocol was used with following monoclonal antibodies: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD23, CD34, CD38, CD45, CD56, CD200, FMC7, αβ TCR, γδ TCR, kappa light chains, and lambda light chains in different tube combinations. Briefly, 100-300 μl of BMA/PB samples was incubated with antibody cocktails for 20-30 min in dark. It was then washed with phosphate-buffered saline to remove the excess of unbound antibodies. This was followed by red cell lysis using commercial lysing agents, followed by wash and a final suspension of cell pellet in paraformaldehyde. The processed samples were acquired on a BD FACS Canto II platform with FACS Diva software (BD Biosciences, San Jose, CA, USA), which was also used for the analysis of samples.
10
Cell Cycle Analysis of HK-2 Cells
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The cell cycle assay was performed using Cell Cycle and Apoptosis Analysis Kit (Beyotime Biotechnology, C1052) as described previously.24 Briefly, HK‐2 cells were harvested and fixed in cold 70% ethanol at 4°C overnight. The cells were washed with cold PBS and then incubated with propidium iodide (PI) staining buffer at 37°C for 30 min in the dark, subsequently analysed by FACS Canto II platform (BD, FACS Canto II, San Jose, CA, USA).
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