THP-1 cells were seeded at a density of 10.000 c/w in 24-well plates (Sarstedt, Nürnbrecht, Germany). Cells were treated with BTZ and/or HDACI for the indicated time. Supernatant was removed after a centrifugation step and the cells were lysed in 500 µL hypotonic lysis buffer (0.1% TritonX-100, 100 µg/mL Propidium iodide) at 4 °C in the dark overnight. Triton X-100 was obtained from AppliChem (Darmstadt, Germany). Propidium iodide was delivered by Santa Cruz Biotechnology (Heidelberg, Germany). The percentage of apoptotic nuclei with DNA content in sub-G1 was analyzed by flow cytometry using the CyFlow instrument (Partec, Norderstedt, Germany).
Propidium iodide
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany
Propidium iodide is a fluorescent dye used in flow cytometry and microscopy applications. It binds to DNA and is commonly used to stain and quantify cellular DNA content.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Rnase a, by Roche (3 mentions)
Facscanto, by BD (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Modfit lt, by Verity Software House (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Rnase a, by Santa Cruz Biotechnology (2 mentions)
Annexin 5, by Santa Cruz Biotechnology (2 mentions)
Phospho histone h3 serine 10 antibody, by Cell Signaling Technology (2 mentions)
Alexa fluor 488 dc28, by Cell Signaling Technology (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Fluorescein isothiocyanate conjugated goat anti rabbit antibody, by Santa Cruz Biotechnology (2 mentions)
Fv10i, by Olympus (2 mentions)
Acridine orange, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Fluoro styrylbenzene fsb congo red derivative, by Santa Cruz Biotechnology (2 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Alexa fluor 488 conjugated phospho histone h3 ser10 antibody dc28, by Cell Signaling Technology (2 mentions)
40 protocols using propidium iodide
1
Apoptosis Quantification in THP-1 Cells
2
Cell Cycle Analysis by Flow Cytometry
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Cells were collected, fixed in 70% ethanol, and stored at −20 °C overnight. The cells were washed with PBS and resuspended in PBS containing 100 μg/mL RNase A and 0.1% Triton X-100, and incubated at 37 °C for 1 h. Cells were stained with 5 μg/mL propidium iodide (Santa Cruz) for 15 min in the dark. The DNA content of the cells was analyzed using a FACSCanto instrument (BD Biosciences Immunocytometry Systems). Ten thousand cells were collected for each measurement in a triplicate experiment. The percentage of cells in different phases of the cell cycle was analyzed using ModFit LT (Verity Software House).
3
Multimodal Profiling of Cell Lines
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The MCF7 breast cancer cell line, HEK-293 and HEK-293T embryonic kidney cell lines, and monocytic lymphoma cell line U937 were purchased from ATCC® (Manassas, Virginia, USA). The mouse anti-CD47 mAb B6H12 was either produced and purified from hybridoma HB9771 purchased from ATCC®, or purchased from Santa-Cruz Biotechnology (Dallas, TX, USA). The biotinylated mouse anti-CD47 mAb (clone B6H12.2) was purchased from BioLegend (San Diego, CA, USA). The monoclonal sheep anti-SIRPa antibodies and streptavidin-HRP were purchased from R&D Systems (Minneapolis, MN, USA). The monoclonal anti-M13-HRP antibody was from Thermo (Waltham, MA, USA). All the secondary antibodies were purchased from Santa-Cruz Biotechnology. The recombinant human SIRPa-Fc was from R&D Systems. The human CD47 with His tag was a kind gift from P.M. Chumakov. The streptavidin was purchased from MyBioSource (San Diego, CA, USA). The fluorescent lipophilic dyes DiO and DiL were from Biotium (San Francisco Bay Area, CA, USA). The Annexin V-Alexa 488 was from Invitrogen (Waltham, MA, USA), while the propidium iodide was from Santa-Cruz Biotechnology. The Hoechst 33342 was purchased from Sigma (Burlington, MA, USA). The DiL and DiO dyes were from Vybrant Cell-Labeling Solutions, Invitrogen.
4
Cell Cycle Analysis by Flow Cytometry
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Cells were trypsinized and fixed overnight with 70% ethanol at −20 °C. They were incubated with 50 μg/ml RNase A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS for 30 min at room temperature. Propidium iodide (10 μg/ml, Santa Cruz Biotechnology) was added, and the DNA content of the cells was analyzed on a FACSCanto instrument (Becton Dickinson, San Jose, CA, USA). The percentages of cells in different phases of the cell cycle were measured by using ModFit (Verity Software House, Topsham, ME, USA).
5
Cell Cycle and Apoptosis Analysis via Flow Cytometry
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To assess the cell cycle profile, the cells were treated with butein and were subsequently fixed in 95% ethanol with 0.5 % Tween-20 at −20°C overnight. The fixed cells were stained with 50 µg/ml propidium iodide (Santa Cruz Biotechnology, Inc.). For apoptotic cell death, the cells were treated with butein and subsequently stained with annexin V and 7-aminoactinomycin D (7-AAD; Santa Cruz Biotechnology, Inc.). The cells were analyzed using a FACSCalibur (BD Biosciences, San Jose, CA, USA) with CellQuest Pro, version 5.2 software.
6
Phospho-histone H3 Cell Cycle Analysis
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Cells were harvested from a single well of a 6-well plate using trypsin and washed 1x in PBS before fixation with dropwise addition of ice-cold 70% ethanol. After fixation on ice (or storage at −20°C) cells were washed 1x in PBS and permeabilized in 0.25% Triton X-100/PBS solution on ice for 15 minutes. Cells were washed 1x in PBS and resuspended in 1% bovine serum albumin/PBS solution containing 1µl/sample of Phospho-histone H3 (Serine 10) antibody conjugated to Alexa Fluor 488 (DC28, Cell Signaling). After washing 1x in PBS cells were resuspended in a solution of PBS containing 50µg/mL Propidium iodide (Santa Cruz Biotechnology) and 100µg/mL RNAse A (Roche). Flow cytometry was performed on a FACSCalibur (BD Biosciences) and analysed using FlowJo software. Single cells and G1/S/G2 peaks were manually gated. The gates for phospho-H3 positive cells were chosen by comparison to a population in which the antibody was omitted during processing.
7
Gemcitabine and EGFR Inhibitor Combination Therapy
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Gemcitabine, propidium iodide, anti-RRM1, anti-RRM2 (Santa Cruz Biotechnology, Inc., Dallas, TX); DMEM, RPMI 1649 medium, fetal bovine serum (HyClone Laboratories, Inc., South Logan, UT); human RRM1 and RRM2 produced from HEK293 cells (OriGene Technologies, Inc., Rockville, MD); anti-EGFR, anti-GAPDH, DNA damage antibody sampler kit (Cell Signaling Technology, Boston, MA); C18 Zip-tip, Amicon Ultra-15 centrifugal filters, anti-phospho-Histone H2AX (Ser 139) (α-γ-H2AX) (Merck Millipore, Darmstadt, Germany); protein A-sepharose fast flow (GE Healthcare, Chicago, IL ); afatinib, canertinib, dacomitinib, neratinib, erlotinib (LC Laboratories, Woburn, MA) were purchased from manufacturers indicated in parentheses. Other chemicals were mostly from Sigma-Aldrich Corporation (St. Louis, MO).
8
Cell Cycle Analysis by Flow Cytometry
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Flow cytometry was performed following DNA staining with propidium iodide (PI; Santa Cruz Biotechnology, Inc.), according to the manufacturer’s instructions. Briefly, the cells were harvested, washed with phosphate-buffered saline (PBS) and treated with 100 mg/ml RNase A (Santa Cruz Biotechnology, Inc.) for 30 min at room temperature. The cells were then stained with PI (1 mg/ml) solution at 4°C and incubated in the dark for 30 min. The cell cycle distribution was evaluated using a BD FACSArray™ Bioanalyzer system (BD Biosciences, San Jose, CA, USA).
9
Immunofluorescence Staining of GPR30 and p-eNOS in HUVECs
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The immunofluorescence staining of GPR30 and p-eNOS in the HUVECs was performed as previously described (23 (link)). Following treatment, the HUVECs were fixed in 4% formaldehyde (Aladdin, Shanghai, China) and blocked with 10% normal goat serum (Sigma-Aldrich), then incubated with anti-GPR30 antibody (1:80 dilution) or anti-p-eNOS Ser1177 antibody (1:25 dilution). A fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (1:50 dilution; sc-2012; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was then used. The nuclei were stained with propidium iodide (3 mg/ml; Santa Cruz Biotechnology, Inc.). Images were acquired using a confocal microscope (FV10i; Olympus Corp., Tokyo, Japan).
10
Cell Cycle Analysis by Flow Cytometry
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Cells were collected by trypsinization from a 70%–80% confluent 10-cm dish. The pellet was suspended in 300 µL of PBS, and 700 µL of 100% chilled ethanol was added dropwise while simultaneously vortexing the cells. The cells were fixed at least overnight at −20°C. At the time of staining, cells were pelleted and washed twice with PBS. The pellet was then resuspended in 250 µL of PBS containing 250 µg/mL RNase A (Roche) and incubated for 30 min at 37°C. PBS (250 µL) containing 50 µg/mL propidium iodide (Santa Cruz Biotechnology) was then added, and cells were incubated in the dark for 10 min at room temperature. Data for at least 10,000 live cells were collected using FACSCalibur (BD Biosciences). Analysis was performed using the FlowJo software. A double-discrimination gate was used to exclude doublets, and G0/G1, S, and G2 percentages were calculated using the Watson pragmatic algorithm. Cells >4N were manually gated.
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