The present study established the following experimental groups: Control, cells + Hoechst 3342 (n=9 samples from 9 patients); and drug-treated, cells + verapamil (Sigma-Aldrich) + Hoechst 33342 (n=9). Cells in staining medium (106 cells/ml DMEM with 10% FCS) were labeled with Hoechst 33342-bis-benzimide stock (final incubation concentration, 5 µl/ml; Sigma-Aldrich) and optionally treated with verapamil (final incubation concentration, 0.8 µl/ml). The cells were mixed and incubated in a water bath at 37°C for 90 min. The cells were then centrifuged (1,800 × g for 10 min at 4°C) and re-suspended in 500 µl Hank's balanced salt solution (Sigma-Aldrich) containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Sigma-Aldrich). Finally, the cells were counterstained with propidium iodide (PI; Sigma-Aldrich) 2 µg/ml at 4°C. Cells were filtered through a 50-µm nylon mesh (BD Biosciences, Franklin Lakes, NJ, USA) to remove cell clumps and filled into labeled FACS tubes. Separate tubes with medium (DMEM with 10% FCS) were kept for sterile sorting of SP cells and main population cells. The cells were sorted using a flow cytometer (FACS Aria II; BD Biosciences). The Hoechst 33342 dye was excited at 355 nm and its dual-wavelength fluorescence was analyzed (blue, 450 nm; red, 675 nm).
Verapamil
Manufactured by Merck Group
Sourced in United States, Germany, France, United Kingdom, China, Japan, Spain, Italy, Sao Tome and Principe, Switzerland, Australia, Ireland, Belgium
Verapamil is a laboratory product manufactured by Merck Group. It is a calcium channel blocker that inhibits the movement of calcium ions through cell membranes, which can affect various physiological processes. The core function of Verapamil is to serve as a research tool for the study of calcium-dependent mechanisms in biological systems.
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Lab products found in correlation
Hoechst 33342, by Merck Group (107 mentions)
Propidium iodide, by Merck Group (48 mentions)
Rhodamine 123, by Merck Group (46 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (44 mentions)
Doxorubicin, by Merck Group (43 mentions)
Paclitaxel, by Merck Group (39 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (37 mentions)
Cisplatin, by Merck Group (34 mentions)
Hoechst 33342, by Thermo Fisher Scientific (29 mentions)
Vincristine, by Merck Group (28 mentions)
Hoechst 33342 dye, by Merck Group (26 mentions)
Nifedipine, by Merck Group (24 mentions)
Trypsin edta, by Thermo Fisher Scientific (24 mentions)
Triton x 100, by Merck Group (19 mentions)
Probenecid, by Merck Group (17 mentions)
Mitoxantrone, by Merck Group (16 mentions)
Ko143, by Merck Group (14 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (14 mentions)
Quinidine, by Merck Group (13 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (13 mentions)
698 protocols using verapamil
1
Hoechst 33342 Side Population Cell Isolation
2
Cytotoxicity Evaluation of Verapamil and Amphotericin B
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The calcium channel blocker verapamil (Sigma-Aldrich, St Louis, MO) and the antifungal amphotericin B (Sigma-Aldrich, St Louis, MO) were used in this study. Stock solutions were prepared in dimethyl sulfoxide (DMSO) and subsequently diluted in Roswell Park Memorial Institute 1640 (RPMI 1640) medium. The maximum DMSO concentration in the final medium was 5%.
Evaluation of cell viability (in vitro cytotoxicity) using Resazurin Evaluation of cell viability was performed using the Resazurin method. For this assay, the HaCaT cell line (human keratinocytes) was purchased from the Rio de Janeiro Cell Bank (BCRJ) and was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 2% antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin). HaCaT cells were seeded at a concentration of 10 6 cells/mL in a 96-well microplate and incubated for 24 h at 37 °C with 5% CO 2 . Cells were treated with verapamil concentrations ranging from 2.4 to 1250 μM for 24 h. Resazurin (Sigma-Aldrich, St Louis, MO) was used to measure cell viability as described earlier (Pavan et al. 2010) . According to ISO 10993-5 (2009) , drug concentrations provoking a >30% reduction in cell viability are considered cytotoxic.
Evaluation of cell viability (in vitro cytotoxicity) using Resazurin Evaluation of cell viability was performed using the Resazurin method. For this assay, the HaCaT cell line (human keratinocytes) was purchased from the Rio de Janeiro Cell Bank (BCRJ) and was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 2% antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin). HaCaT cells were seeded at a concentration of 10 6 cells/mL in a 96-well microplate and incubated for 24 h at 37 °C with 5% CO 2 . Cells were treated with verapamil concentrations ranging from 2.4 to 1250 μM for 24 h. Resazurin (Sigma-Aldrich, St Louis, MO) was used to measure cell viability as described earlier (Pavan et al. 2010) . According to ISO 10993-5 (2009) , drug concentrations provoking a >30% reduction in cell viability are considered cytotoxic.
3
Hoechst 33342 staining for side population
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Single-cell suspensions of cells were detached from dishes with trypsin-EDTA (Invitrogen) and suspended at 106 cells/ml in HBSS supplemented with 3% FCS and 10 mM Hepes. These cells were then incubated at 37°C for 90 min with 20 µg/ml Hoechst 33342 (Sigma-Aldrich), either alone or in the presence of 100 nM/ml verapamil (Sigma-Aldrich), an inhibitor of the verapamil-sensitive ABC transporter. After 90-min incubation, the cells were centrifuged immediately for 5 min at 300 g and 4°C and resuspended in ice-cold HBSS. The cells were kept on ice to inhibit efflux of the Hoechst dye, and 1 µg/ml propidium iodide (Sigma-Aldrich) was added to discriminate dead cells. Finally, these cells were filtered through a 40-µm cell strainer (Falcon; BD) to obtain single-suspension cells. Cell dual-wavelength analysis and purification were performed on a dual-laser FACS (AriaIII; BD). Hoechst 33342 was excited at 355 nm UV light and emitted blue fluorescence with a 450/20 band-pass filter and red fluorescence with a 670-nm edge filter long-pass. propidium iodide–positive (dead) cells were excluded from the analysis.
4
Macrophage Cytokine Response to Antibiotics
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Monocytes were resuspended at a density of 1 × 106/ml in medium as described above. The cell suspension was seeded on 96-well plates, and azithromycin, clarithromycin, or roxithromycin (all obtained from Sigma) were added in the indicated concentrations just before stimulation with either 100 ng/ml LPS 0111:B4 (L-4391, Sigma) or 100 ng/ml flagellin (Invivogen, San Diego, CA). After 18–20 h incubation, the supernatants were collected, centrifuged for 5 min at 400 × g and used for further analysis. In the Ca++-depleting experiments, cells were treated as described above but were stimulated in medium containing 10 mM EGTA (Sigma). In the Ca++-channel blocking experiments, cells were incubated with 125 μM verapamil (Sigma) for 30 min before treatment with azithromycin and LPS stimulation (verapamil was not removed during stimulation). Cytokine measurements of TNF-α, IL-1β, IL-1α, IL-6, and IL-8, were performed by Luminex® testing using specific matched-pair antibodies and recombinant cytokines as standards (Merck Millipore, Billerica, MA). IL-18 was measured with a human IL-18 Instant ELISA (eBioscience, Vienna, Austria; detection limit: 9.2 pg/ml).
5
Modulation of Mx1-Cre Induction Pathways
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Poly I:C (Invivogen) was administered intraperitoneally (i.p.) every other day at 5 mg/kg for three doses for Mx1-Cre induction. NAC (Sigma #A7250) was administered at 50 mg/kg in PBS i.p. daily and supplemented at 1 mg/ml via drinking water which was refreshed twice a week. CFZ (CFZ/PR-171, Selleckchem), rapamycin (sirolimus, Selleckchem) and verapamil (Sigma #V4629) were dissolved in ethanol first and then diluted in 1% PEG400/1%Tween80/PBS for injections. CFZ was injected intravenously at 2 mg/kg on days 1 and 2 of each week. rapamycin was administered at 4 mg/kg i.p. daily and supplemented at 15 ng/ml in drinking water when specified50 (link). RTKi PKC412 (Midostaurin) was purchased from Selleck Chemicals (Cat# S8064) and injected i.p. daily at 3 mg/kg. LiCl40 (link) (1 mEq/kg, Fisher #L121) and verapamil41 (link) (25 mg/kg, Sigma #V4629) were also injected i.p. daily. All drugs used for rescue purpose were administered starting the day after the pIpC induction and throughout the course until the day of analysis.
6
Rhodamine 123 Efflux Assay for MDR1 Activity
7
Isolation and Analysis of Side Population Cells
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To identify and isolate side population (SP) cells, the cells were dissociated and resuspended at 1 × 106 cells/ml in DMEM supplemented with 5% fetal bovine serum, preincubated at 37°C for 30 minutes without or with 100 mM verapamil (Sigma). If verapamil was used in the subsequent steps, it was included at 50 mM. Cells were labeled with 2.5 mg/ml Hoechst 33342 (Sigma) in staining media at 37°C for 90 minutes, incubated on ice for 10 minutes, washed twice with ice-cold phosphate-buffered saline (PBS), and then analyzed (20,000 cells per experiment with three replication) on a FACStar plus (BDIS) cell sorter equipped with dual Coherent I-90 lasers.
8
Fluorescence-based Assay for Efflux and Uptake
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Ethidium bromide (EthBr) accumulation/efflux and Nile red uptake were measured by fluorescence intensity, as previously described (Chuang et al., 2015 (link)) with minor modifications. Briefly, mid-log-phase or nutrient-starved cultures were washed with PBS and then stained with 2 μg/ml EthBr (Sigma) or with 0.125 μM Nile red (Sigma). For the EthBr accumulation assay with efflux inhibitor, verapamil (Sigma) was added and bacteria incubated with 2 μg/ml EthBr and 100 μg/ml verapamil for 60 min. In all assays, the cells were incubated in 96-well plates, and analysis was performed at the indicated time points by excitation at 544 nm and emission at 590 nm on a FLUOstar OPTIMA microplate reader (BMG LABTECH). All data were normalized to the time zero reading of each well and to bacterial density. All experiments were performed in triplicate and repeated at least twice, yielding similar results.
9
Rhodamine 123 Efflux Assay for MDR1 Activity
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PBMC were loaded with 10µg/ml Rhodamine 123 (Rh123; Sigma, Dorset, UK) in RPMI + 1% Bovine Serum Albumin (BSA) for 1hr on ice. Cells were resuspended in R10 with or without inhibitors, and either kept on ice (loading control) or cultured at 37°C (efflux) for 30mins. MDR1 inhibitors cyclosporin A (CsA; Sigma) and verapamil hydrochloride (verapamil; Sigma) were added at the lowest inhibitory concentration; 0.5µM and 50µM, respectively. After efflux, cells were returned to ice and surface stained for analysis. Rh123 fluorescence was collected at 585/40nm. Efflux was calculated as:
(GeoMFI fluorescent substrate)Loading control − (GeoMFI fluorescent substrate)Efflux /(GeoMFI fluorescent substrate)Loading control
10
Hoechst 33342 and Verapamil Labeling of Cancer Stem Cells
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H1650 cells were trypsinized and pretreated with Dulbecco's modified Eagle medium containing 2% fetal calf serum (staining medium) for 10 min at 37°C. The cells were labeled in the same medium at 37°C for 90 min with 2.5 μg/ml Hoechst 33342 dye (molecular probes), either alone or in combination with 50 μM of verapamil (Sigma), which is an inhibitor of several verapamil-sensitive ATP-binding cassette (ABC) transporters.[ 25 ] Finally, cells were counterstained with 1 μg/ml of propidium iodide to label dead cells. Then, 3-5 × 10 4 cells were analyzed in a FACSVantage fluorescence-activated cell sorter (Becton Dickinson, USA) using a dual wavelength analysis (blue, 424-444 nm; red, 675 nm) after excitation with 350 nm ultraviolet light. Propidium iodide-positive dead cells (<15%) were excluded from the analysis. The SP cells or non-SP (NSP) cells were sorted for reverse transcription polymerase chain reaction (RT-PCR) and xenograft assays.
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