As previously described by Gagnaire [44 ], hemocyte mortality was measured in 200 μL of cell suspension sampled from each condition (3 replicates) and at each sampling time. Cells were incubated in the dark for 30 min at 4°C with 10 μL of propidium iodide (ThermoFisher Scientific, P3566; PI). PI is a fluorescent DNA/RNA-specific dye that only permeates the membranes of dead cells and stains their nucleic acids. Cells that emit red fluorescent light (FL3: 560–670 nm) when stained by PI were thus considered to be dead.
P3566
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The P3566 is a high-performance laboratory centrifuge designed for a variety of applications. It features a robust and durable construction, ensuring reliable and consistent performance. The centrifuge can accommodate multiple rotor types and sample volumes, enabling versatile usage across different laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (7 mentions)
Propidium iodide, by Thermo Fisher Scientific (6 mentions)
C3100mp, by Thermo Fisher Scientific (5 mentions)
Facscalibur, by BD (4 mentions)
Ls004188, by Worthington (3 mentions)
Rnase a, by Merck Group (3 mentions)
Fm4 64, by Thermo Fisher Scientific (3 mentions)
A1r confocal microscope, by Nikon (3 mentions)
Flow check fluorospheres, by Beckman Coulter (3 mentions)
Facs sortware 1, by BD (3 mentions)
Influx v7 sorter, by BD (3 mentions)
Bz x700 microscope, by Keyence (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Cytoflex flow cytometer, by Beckman Coulter (2 mentions)
Cytexpert software, by Beckman Coulter (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Dmi8 inverted microscope, by Leica (2 mentions)
H3570, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (2 mentions)
Specific anti brdu fitc monoclonal antibody, by BD (2 mentions)
116 protocols using p3566
1
Measuring Hemocyte Mortality with PI Staining
2
Germ Cell Sorting and Purification
3
Calcium Flux Imaging of Microbubble-Mediated Sonoporation
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To image calcium flux, the spheroids were loaded with freshly dissolved, cell-permeable 1 µM Fluo 4-AM (F14201, Thermo Fisher Scientific), a green fluorescent, intracellular Ca2+ indicator, for 30 min at 37 °C. Then, the spheroids were moved to a glass bottom dish (P50G-0-30-F, MatTek, Ashland, MA, USA) containing media with 1 × 106 microbubbles. A coverslip was placed above the spheroids. The presence of microbubbles on the spheroids was microscopically confirmed. Using an in-house designed cone housing and a water immersion transducer (A303S-SU, 0.5-inch diameter, Olympus NDT), US was delivered (frequency 1 MHz; PNP 250 kPa; pulse length 10 µs; pulse interval 10 ms; treatment duration 10 s), and green fluorescence was measured for up to 1 min (Supplementary Videos S3 , 4 ). The involvement of mechanosensitive channels in UTMC-induced Ca2+ influx was confirmed by pretreating spheroids with 1 µM GsMTx4 (STG-100, Alomone labs, Jerusalem, Israel) for 30 min. The occurrence of sonoporation was confirmed by loading spheroids with 120 µM PI (P3566, Thermo Fisher Scientific) in the same setup (Supplementary Video S2 ). The videos were quantified using Fiji.
4
Germ Cell Sorting and Purification
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Germ cell sorting was as described (Gainetdinov et al., 2018 (link); Yu et al., 2022 (link)). Briefly, we de-capsulated the testis specimens and incubated with 1× Gey′s Balanced Salt Solution (GBSS, Sigma, G9779) containing 0.4 mg/ml collagenase type 4 (Worthington; LS004188) at 33°C for 15 min. Thereafter, we washed the seminiferous tubules twice with 1× GBSS, and incubated them with 1× GBSS containing 0.5 mg/ml trypsin and 1 μg/ml DNase I at 33°C for 15 min. Next, the seminiferous tubules were gently pipetted up and down for 3 min through a Pasteur pipette to homogenize at 4°C on. Trypsin was inactivated with fetal bovine serum (FBS; f.c. 7.5% [v/v]), and the cell suspension was passed through a pre-wetted 70 μm cell strainer. Cells were recovered by centrifugation at 300 × g at 4°C for 10 min, resuspended in 1× GBSS containing 5% (v/v) FBS, 1 μg/ml DNase I, and 5 μg/ml Hoechst 33342 (Thermo Fisher, 62249), and incubated at 33°C for 45 min rotating at 150 rpm. Finally, we added propidium iodide (0.2 μg/ml, f.c.; Thermo Fisher, P3566) directly to the cells which were further filtered through a pre-wetted 40 μm cell strainer. Cell sorting (FACS Core, University of Massachusetts Medical School) was performed as described (Bastos et al., 2005 (link); Yu et al., 2022 (link)).
5
Imaging HepG2 Cell Nuclei with PI
6
Cell Cycle Synchronization and FACS Analysis
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siRNA treated U2OS cells were synchronized in G1 phase using a double thymidine block. Specifically, 7.5 × 105 siRNA transfected U2OS cells were treated with 2.5 mM thymidine in media for 19 hr. Cells were washed twice with PBS and incubated in media for 9h, prior to the addition of 2.5 mM thymidine for 16 hr to synchronize cells in G1 (this time point is referred to as ‘0 hr’ in figure S6A). For fluorescence-activated cell sorting (FACS) analysis of cell cycle profile, cells were trypsinized, washed twice in ice-cold PBS and resuspended in 0.5 mL 1X PBS. Cells were fixed by gently vortexing while adding 4.5 mL ice-cold 70% ethanol drop-wise followed by 30 min incubation at 4°C. Fixed cells were washed twice in 1X PBS and resuspended in 0.5 mL Staining Solution (0.1% Triton X-100, 10 μg/mL propidium iodide (P3566, ThermoFisher Scientific), 100 μg/mL RNase A (R5503-100MG, Sigma-Aldrich)) and incubated for 30 min in the dark at room temperature. Cells were analyzed on a BD LSR II Flow Cytometer (BD Biosciences) and profiles generated using FlowJo v10.1 software.
7
CAR T Cell-Mediated Cytotoxicity Assay
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The elimination index was determined according to a previous study. Briefly, CAR T cells were co-cultured with target cells at an E:T ratio of 1:6 in RPMI 1640 fully supplemented in the absence of cytokines. After 48 h, cells were stained with propidium Iodide (Thermo Fisher, P3566), and live/dead cells were analyzed with flow cytometry (BD Biosciences, LSRII). The elimination index was calculated as follows: 1 - (ratio of live target cells with CAR T cells / ratio of live target cells in the control group).
8
Quantifying Cell Viability with Fluorescent Dyes
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We used Propidium iodide (5 µg/ml, Thermo # P3566) (Excitation/Emission: 535/617 nm) for the initial evaluation of damaged/dead cells. LIVE/DEAD™ Viability/Cytotoxicity Kit was used for quantifying cell viability (Invitrogen #L3224). Calcein AM (1 µM) was used for labeling live cells and the Ethidium Homodimer-1 (2 µM) for labeling dead cells. The live and dead cells were quantified by green fluorescent (Excitation/Emission: 485/528 nm) and red fluorescent (Excitation/Emission: 535/617 nm), respectively.
9
Cell Cycle Analysis with PI and RNase
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Cells were harvested with trypsin and re-suspended in 75% ethanol. Pellets were washed twice with PBS and re-suspended in 50 µg/mL propidium iodide (ThermoFisher, P3566) and 5 µg/mL ribonuclease (ThermoFisher, EN0531). Fortessa instrument and ModFit software were used for cell cycle analysis.
10
Propidium Iodide Staining for Cell Death Analysis
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The propidium iodide staining was used to assess cell death. HEK293T cells and HeLa cells were treated with DMSO, JQ-1 (1 μM), I-BET-762 (2 μM), Erastin (20 μM), and Erastin with JQ-1 or I-BET-762 respectively for 48 h. HepG2, RKO, PC3 cells and stable BRD4 knockdown cells were also treated as indicated drugs. Then the cells were treated with 5 ug/mL propidium iodide (P3566, Thermo Fisher Scientific) for 15 min. The samples were photographed to observe cell morphology and death.
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