Medium with a final propidium iodide concentration of 1 μM (stock solution: 1 mM in water) (Krämer et al., 2016 (link)) was used for dead staining (propidium iodide; Cayman Chemical Company, USA). The pH was then adjusted, and the medium was sterile filtered to prevent channel blockage. dMSCC with individual stress pulses of pH 5 for 2, 6 and 9 h and pH 10 for 2 and 6 h were performed. For a more detailed description, see “Setup and Microfluidic Cultivation.”
Propidium iodide
Manufactured by Cayman Chemical
Sourced in United States
Propidium iodide is a fluorescent dye that binds to DNA. It is commonly used in flow cytometry and microscopy applications to detect and quantify DNA content in cells.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Accuri c6 plus, by BD (2 mentions)
Fcs express 6 flow cytometry software, by De Novo Software (1 mentions)
Lsr 2 flow cytometry analyzer, by BD (1 mentions)
4 oht, by Merck Group (1 mentions)
Y 27632, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest pro software, by Olympus (1 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Multitox fluor multiplex cytotoxicity assay, by Promega (1 mentions)
Spark 20m plate reader, by Tecan (1 mentions)
Alexa fluor 488 annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Facs cytometry, by BD (1 mentions)
Fluorescein diacetate, by Merck Group (1 mentions)
Facsdiva software, by BD (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Facs caliber, by BD (1 mentions)
Multicycle for macintosh software, by BD (1 mentions)
In situ apoptosis detection kit, by Takara Bio (1 mentions)
Epigallocatechin gallate (egcg), by Merck Group (1 mentions)
23 protocols using propidium iodide
1
Propidium Iodide Staining and dMSCC Stress Pulses
2
Flow Cytometry Analysis of Cell Treatment
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Flow cytometry analysis was performed as previously described [11 (link),16 (link),39 (link)]. Briefly, MCF-7 and BT474 cells were seeded at a density of 1 × 106 cells/plate and their media was changed two times in every two days. Following an overnight treatment with 4-OHT (10−6 M) (Sigma) and SEL (10−7 M) (Selleckchem) alone and in combination, cells were collected with 0.1% FBS containing 1× PBS, washed, resuspended at 1–2 × 106 cells/mL and fixed with ice cold ethanol for 24 h. Next day, cells were washed with 1× PBS and incubated in 10 ng
RNAase for one hour at room temperature. Cells were stained with 0.25% Propidium Iodide (#10008351, Cayman Chemicals, Ann Arbor, MI, USA) for one hour and results were analyzed by BD™ LSR II Flow cytometry analyzer (BD Biosciences Inc., San Jose, CA, USA). Following analysis, results were analyzed by FCS Express 6 Flow Cytometry Software (DeNovo Software,https://www.denovosoftware.com/site/Flow-RUO-Overview.shtml ) and all experiments were repeated three times.
RNAase for one hour at room temperature. Cells were stained with 0.25% Propidium Iodide (#10008351, Cayman Chemicals, Ann Arbor, MI, USA) for one hour and results were analyzed by BD™ LSR II Flow cytometry analyzer (BD Biosciences Inc., San Jose, CA, USA). Following analysis, results were analyzed by FCS Express 6 Flow Cytometry Software (DeNovo Software,
3
Multimodal Labeling of Intestinal Crypts
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To label all nuclei in the crypt base, Hoechst-33342 (Thermo Fisher, Watham, MA; 2 mg/kg) was injected under the window 1 hour before imaging. To inhibit ROCK protein signaling, Y-27632 (Sigma Aldrich, St. Luis, MO; 5 mg/kg mouse weight in saline) was injected under the window before the start of imaging. This dosage was selected as the highest dose which did not alter crypt structure or change GFP expression patterns over one day (data not shown). Saline (0.9%) (Phoenix Pharmaceutics, Burlingame, CA) was injected for the sham-treated group. To label damaged cells, propidium iodide (Cayman Chemical Company, Ann Arbor, MI; 100 µl of 1 mg/ml in PBS) was injected retro-orbitally.
4
Cell Cycle Analysis by Flow Cytometry
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Cells were cultured in 6-well plates as described above. After 72 h incubation, cells with approximately 80% confluence were collected with trypsin. The cells were washed in PBS and fixed in ice-cold 70% ethanol for 30 min at −20 °C. The fixed cells were washed three times and suspended in PBS containing 50 μg/mL of RNase A for 30 min, then incubated with 50 μg/mL propidium iodide (PI; Cayman Chemical, Ann Arbor, MI, USA) for 30 min in the dark. Subsequently, the samples were acquired in a FACSCalibur (Becton Dickinson, San Diego, CA, USA) system and PI incorporation was estimated using CellQuest Pro software (Olympus, Tokyo, Japan). For each measurement, at least 25,000 cells were acquired. An analysis of the cell cycle was performed using the Watson modelling algorithm from the FlowJo software (Ashland, OR, USA).
5
Cell Cycle Analysis of AuNP Treatments
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The 200,000 cells were seeded on each well of the 12-well plate. After 24h, the medium was removed, and a solution of culture medium with each AuNP, RGD-PEG800-AuNPs10nm, RGD-PEG2000-AuNPs10nm, RGD-PEG800-AuNPs30nm, and RGD-PEG2000-AuNPs30nm, at a concentration of 0.0004, 0.0008 and 0.0012 mg/mL was added to cells and incubated for 24, 48, 72 h. Afterward, cells were collected and fixed in 70% ice-cold ethanol solution (POCh, Poland). Samples were stored at −20°C. For the cell cycle analysis, the cell pellet was dyed using 1 mg/mL propidium iodide (Cayman Chemical Company, USA) and 10 mg/mL RNase I enzyme (PanReac AppliChem, ITW Reagents, Chicago, IL, USA) in PBS solution. Cells were incubated for 30 min at 37 °C, protected from light. Fluorescence was measured at 611 nm. The obtained results were analyzed using FlowJo v10.
6
Nanorod Cytotoxicity Evaluation in Cells
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A total of 300,000 cells per well were seeded in a 6-well plate. After 24 h, the medium was removed, and a solution of medium and nanorods was added at the final concentrations of 2, 4, 6, 8, and 10 μM in a volume of 2 mL per well. After 4, 6, and 24 h, cells were collected and fixed in 70% ethanol solution (POCh, 44101 Gliwice, Poland). The prepared samples were stored at −20 °C for further analysis. The pellet was suspended in 200 μL of a mixture consisting of 10 μL of 1 mg/mL propidium iodide (Cayman Chemical Company, Ann Arbor, MI, USA), 2 μL of 10 mg/mL RNase I enzyme (PanReac AppliChem, ITW Reagents, Chicago, IL, USA), and 188 μL of PBS solution. Samples were incubated for 30 min at 37 °C, protected from light. Fluorescence was measured at 611 nm. The obtained results were analyzed using FlowJo v10.
7
Fungal Spore Microscopy Protocol
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About 1 × 103 conidia were incubated overnight in liquid medium on glass or polypropylene carriers. Differential interference contrast, bright-field, and fluorescence images were captured using Olympus BH-2, IX71, and BX51 microscopes and were adjusted for brightness and contrast with ImageJ. To stain dead cells, cultures were incubated for 5 min with 10 μM propidium iodide (PI; Cayman Chemicals) prior to imaging.
8
Antibody-mediated Neurotoxicity in Nodding Syndrome
9
Quantifying Growth Factor Starvation-Induced Cell Death
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To investigate growth factor starvation‐induced cell death, cells were plated in a 12‐well dish and serum starved in the presence or absence of 20 ng/ml EGF. Twenty‐four hours later, propidium iodide (1 μg/ml, #10008351, Cayman) was added to cells and analysed using a Tecan Spark20M plate reader. Cells were then fixed with 3.7% PFA and total cell number was obtained by staining with DAPI and measurement using the Tecan plate reader. The ratio of propidium iodide/DAPI relative fluorescence was then calculated to obtain relative values of cell death. Representative cell images were taken on a Leica DM IL LED microscope using Qimaging Retiga EXi Fast1394.
10
Cell Cycle and Apoptosis Analysis
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After treating NSCLC cells with KPT-185 for 48 h, they were stained with propidium iodide (PI) staining buffer (Cayman Chemical, Ann Arbor, MI, USA) for 30 min at room temperature and then measured by FACS cytometry (BD Biosciences, NJ, USA). The DNA histograms were analyzed using ModFit LT cell cycle analysis software (Verify Software House).
Cell apoptosis was detected with an Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen) according to the kit instructions. Briefly, after treating with KPT-185 for 48 h, the cells from both suspension and adherence were collected and co-incubated with Annexin V-fluorescein isothiocynate (FITC) and PI, then measured by FACS cytometry (BD Biosciences). The percentage of Annexin V and PI negative cells was determined based on the dot plots of FITC and PI.
Cell apoptosis was detected with an Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen) according to the kit instructions. Briefly, after treating with KPT-185 for 48 h, the cells from both suspension and adherence were collected and co-incubated with Annexin V-fluorescein isothiocynate (FITC) and PI, then measured by FACS cytometry (BD Biosciences). The percentage of Annexin V and PI negative cells was determined based on the dot plots of FITC and PI.
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