Verapamil hydrochloride, PLGA (Resomer RG 504 H) and Polyvinyl alcohol (Mowiol 4-88) were procured from Sigma Aldrich (USA). Doxorubicin Hydrochloride was a gift from Deva, Turkey. Annexin V-FITC and propidium iodide were obtained from Serva, Germany. All other reagents and chemicals used were of analytical grade.
Propidium iodide
Manufactured by Serva Electrophoresis
Sourced in Germany
Propidium iodide is a fluorescent dye used in molecular biology for nucleic acid staining. It binds to DNA by intercalating between the bases. Propidium iodide is commonly used for cell cycle analysis and to detect cell death or apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Rnase a, by Qiagen (2 mentions)
Facscalibur, by BD (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Verapamil hydrochloride, by Merck Group (1 mentions)
Polyvinyl alcohol mowiol 4 88, by Merck Group (1 mentions)
Idasanutlin, by Selleck Chemicals (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Fitc conjugated anti brdu antibody, by BioLegend (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Modfit lt software, by Verity Software House (1 mentions)
Digitonin, by Serva Electrophoresis (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Cell strainer cap, by BD (1 mentions)
Cystain pi absolute p, by Sysmex (1 mentions)
Rnase a, by Serva Electrophoresis (1 mentions)
Dimethyl sulfoxide (dmso), by Serva Electrophoresis (1 mentions)
Acridine orange, by Merck Group (1 mentions)
11 protocols using propidium iodide
1
Doxorubicin-Loaded PLGA Nanoparticles
2
Cell Cycle Analysis with BrdU Labeling
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The cells were treated with DMSO or idasanutlin (RG7388, Selleck Chemicals, Houston, TX, USA) for 24 h with pulse-labeling using 10 µM bromodeoxyuridine (BrdU, Sigma Aldrich) for the last hour of the treatment. After that, the cells were harvested by trypsinization, fixed with 96% ethanol and stored at −20 °C. The cells were centrifuged and suspended in 2 M HCl/0.5% Triton X-100, added dropwise. Following 30 min incubation at room temperature and centrifugation, the cells were resuspended in 0.1 M Na2B4O7, pH 8.5, centrifuged again and suspended in 0.5% Tween 20/1% BSA/PBS. FITC-conjugated anti-BrdU antibody (BioLegend, cat. 364104, San Diego, CA, USA) was then added (5 µL per 106 cells) and the cells were incubated at 4 °C overnight with gentle shaking (200 rpm). The cells were centrifuged, suspended in PBS containing 5 µg/mL of propidium iodide (PI, SERVA Electrophoresis GmbH, Heidelberg, Germany) and 10 µg/mL RNase A (Thermo Scientific, Waltham, MA, USA), incubated for 20 min at room temperature, and analyzed with BD FACSVerse flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Cell cycle distribution was analyzed using ModFit LT Software (Verity Software House, Topsham, ME, USA).
3
Assessing Cell Viability via Propidium Iodide
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To ascertain the viability of the cells in the wells, a propidium iodide / digitonin assay was used. Cells were detached with 0.05% trypsin-EDTA (Gibco, Carlsbad, CA, USA, 15400) and centrifuged together with the original supernatant, in PBS resuspended, again centrifuged, resuspended and mixed with 5 µM propidium iodide (Invitrogen, Carlsbad, CA, USA, P3566) to stain dead cells, distributed in a black 96-well plate and incubated for 20 min at 37 °C. The fluorescent signal was measured in a plate reader (Synergy HT, BioTek, Bad Friedrichshall, Germany) at an excitation of 485/20 and an emission of 645/40. Afterwards every living cell was destroyed with 600 µM digitonin (Serva, Heidelberg, Germany, 19551) and again after an incubation time of 20 min at 37 °C, the propidium iodide signal was measured. To determine the number of living cells, the corrected values from first measurement were subtracted from those of second measurement using Excel (control: PBS plus propidium iodide, then digitonin).
4
Ploidy Level Analysis by Flow Cytometry
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Measuring of relative DNA amount of nuclei occurred by flow cytometry (Facs calibur, Becton Dickinson, BD) with a red fluorescence laser as basis for detection of ploidy level. For each probe, leaf material was chopped with razor blades in 500 μl nuclei extraction buffer (CyStain PI absolute P, Sysmex) and stained with the corresponding staining buffer, containing 5 % polyvinylpyrrolidone 25 (Serva) and 0.6 % propidium iodide (Serva). Immediately after staining, the nuclei suspension was filtered using a 5 ml polystyrene round-button tube with a cell-strainer cap (BD). For reference, radish was measured in separate sample after five samples of balm.
5
WQF 044 Cytostatic Assay Protocol
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Rnase A was from Qiagen (Hilden, Germany). Propidium iodide (50 µg/ml) was from Serva (Heidelberg, Germany). Further the zVAD-fmk (pancaspase/ panprotease) inhibitor was used. All common cytostatics were provided by the Children’s Hospital of the City Cologne, Amsterdamer Straße. The cytostatic agents were freshly dissolved as stock solutions in DMSO prior to the experiments. They were diluted with the respective cell culture media or buffer during the assay procedures. The substance WQF 044 was dissolved in a 40-mM stock solution of DMSO. Next to the regular control cells in the experiments, some cells were incubated with an equal amount of DMSO only, as DMSO control. The results showed similar effects to those obtained in the untreated controls.
WQF 044 was synthesized and characterized according to Berkessel et al. (2007 (link)).
WQF 044 was synthesized and characterized according to Berkessel et al. (2007 (link)).
6
Organoarsenic Compounds: Cell Viability Assay
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Propidium iodide (50 µg/mL) and RNase A were obtained from Serva (Heidelberg, Germany) and Qiagen (Hilden, Germany), respectively. Common cytostatics were provided by the Children’s Hospital Cologne Amsterdamer Straße and the pharmacy of the Hospital Cologne Merheim, Kliniken der Stadt Köln (Cologne, Germany). The molecular structures of the investigated organoarsenic compounds As1 to As11 are listed in Table 1 . The synthesis of these materials was described previously [18 (link)]. All drugs and compounds were dissolved in DMSO (Serva, Heidelberg, Germany) prior to the treatment of different cell lines.
7
Dissolved Drugs and Compound Preparation
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RNase A was from Qiagen (Hilden, Germany), propidium iodide (50 µg/ml) from Serva (Heidelberg, Germany), and doxorubicine (Doxo), vincristine (Vcr) and cytarabine (AraC) were provided by the Charité, Berlin, Germany. Drugs were freshly dissolved in dimethylsulfoxide (DMSO) prior to the experiments and diluted with the appropriate medium or buffer during the assay procedures. Tris(1,10-phenanthroline)tris(thiocyanato-κN)lanthanum(III) (KP772) was prepared at the Institute of Inorganic Chemistry, University of Vienna, as described previously [14 (link)]. For each experiment, KP772 was freshly dissolved in 0.4% NaCl solution to give a 1 mM stock solution.
8
Quantifying Bacterial Biofilm Viability
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The test specimens were longitudinally split, washed with PBS to remove all canal content, and then stained with acridine orange (SIGMA-ALDRICH, St. Louis, MO, USA) and propidium iodide (SERVA Electrophoresis GmbH, Heidelberg, Germany) to be observed under CLSM (LSM 710, Carl Zeiss Microscopy GmbH, Jena, Germany) at ×20 magnification. In this process, 2–3 random circular areas of 200 starting from the root canal space toward the cementum side were scanned separately. The mean intensity value of red fluorescence (dead bacteria) at the maximum intensity projection of 3-dimensional images was measured using ZEN 2012 blue edition software (Carl Zeiss Microscopy GmbH, Jena, Germany). The mean intensity value of red fluorescence to total fluorescence showed the extent of dead cells in the biofilm.
The mean and standard deviation values for CFUs and CLSM measurements were calculated. The data showed a parametric (normal) distribution by the Kolmogorov-Smirnov and Shapiro-Wilk tests. Comparisons were carried out using 1-way analysis of variance, the post-hoc Tukey test, and the simple t-test. The statistical assessment was conducted using SPSS version 20.0 (IBM Corp., Armonk, NY, USA) with statistical significance indicated by p ≤ 0.05.
The mean and standard deviation values for CFUs and CLSM measurements were calculated. The data showed a parametric (normal) distribution by the Kolmogorov-Smirnov and Shapiro-Wilk tests. Comparisons were carried out using 1-way analysis of variance, the post-hoc Tukey test, and the simple t-test. The statistical assessment was conducted using SPSS version 20.0 (IBM Corp., Armonk, NY, USA) with statistical significance indicated by p ≤ 0.05.
9
Cytotoxicity and Apoptosis Assays in CHO and DLD-1 Cells
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CHO and DLD-1 cell lines
were purchased from
ATCC. Dulbecco Modified Medium (DMEM; Biological Industries), Fetal
Bovin Serum (FBS; Biological Industries),l -glutamine, and
Penicillin/Streptomycin were used as complete cell culture mediums.
Cells were removed from the culture dishes and washed with trypsin-EDTA
(trypsin-ethylenediamine tetraacetic acid) and phosphate buffer (PBS).
Cells were counted using trypan blue. In a cytotoxicity test, cell
viability was determined by using MTT, a tetrazolium salt. In dual
staining, apoptosis/necrosis ratios were determined using Ribonuclease-A,
Propidium Iodide (Serva, Israel), and Hoechst 33342 (Serva, Israel).
All cell culture studies were performed in culture dishes and multiwell
plates (Corning, USA).
were purchased from
ATCC. Dulbecco Modified Medium (DMEM; Biological Industries), Fetal
Bovin Serum (FBS; Biological Industries),
Penicillin/Streptomycin were used as complete cell culture mediums.
Cells were removed from the culture dishes and washed with trypsin-EDTA
(trypsin-ethylenediamine tetraacetic acid) and phosphate buffer (PBS).
Cells were counted using trypan blue. In a cytotoxicity test, cell
viability was determined by using MTT, a tetrazolium salt. In dual
staining, apoptosis/necrosis ratios were determined using Ribonuclease-A,
Propidium Iodide (Serva, Israel), and Hoechst 33342 (Serva, Israel).
All cell culture studies were performed in culture dishes and multiwell
plates (Corning, USA).
10
B-ALL Co-culture Drug Sensitivity Assay
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MSCs were seeded at a density of 4000 cells/well in a 96-well plate in MSC medium 24 h prior to adding B-ALL cells. B-ALL cells were seeded onto MSCs at a density of 1 × 106 cells/mL in SFEM II medium (Stemcell Technologies, Cambridge, UK) supplemented with 20% FCS, 20 ng/mL recombinant IL3 and 10 ng/mL recombinant IL7 (both Peprotech, Hamburg, Germany). After at least 4 h of co-culture, VEN, DAS and DEX were added in combination or as single agents in a concentration range from 1–1000 nM in fixed ratios. After 48 h of drug exposure, non-adherent cells present in supernatant medium were collected, followed by trypsinization and collection of the adherent cell fraction, which contains both MSCs and adhered ALL cells. Cell suspension was stained with human anti-CD19-APC antibody (Biolegend, San Diego, CA, USA) for 20 min at RT and subsequently stained with 1 µg/mL propidium iodide (PI) (Serva, Heidelberg, Germany). Cell viability of CD19-positive cells was assessed via flow cytometry in a FACS Calibur and analyzed with CellQuest Pro software 4.0.2 (both BD Bioscience, San Jose, CA, USA). Relative cell viability was normalized to untreated controls. Drug synergy was analyzed using CompuSyn software 1.0 (BioSoft, Cambridge, UK).
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