BH3 profiling assay was performed as previously described (Fraser et al., 2019 (link)). Briefly, R1/E cells were suspended in 25 μL DTEB buffer (135 mM trehalose, 50 mM KCl, 20 μM EDTA, 20 μM EGTA, 0.1% BSA, 5 mM succinate, 10 mM HEPES-KOH pH 7.5) at 2 × 106 cells/mL, mixed with 25 μL of 4X Dig/Dye (4 mM JC-1, 40 μg/mL oligomycin, 20 mM 2-mercaptoethanol, 0.01% digitonin (w/v) digitonin in DTEB buffer), and stained for 10 min at room temperature. 50 μL of indicated peptide mimics dissolved in DTEB was then added to cells. Fluorescence at 590 nm was monitored using 545 nm excitation on a Varioskan multimode microplate reader (Thermo Fisher Scientific) at 30°C with automated readings every 15 min. The depolarization (%) was quantified as previously described (Ryan and Letai, 2013 (link)).
Varioskan multimode microplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Varioskan multimode microplate reader is a versatile laboratory instrument designed to perform various types of microplate-based assays. It offers a range of detection modes, including absorbance, fluorescence, and luminescence, to enable a wide variety of applications in life science research and clinical diagnostics.
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Lab products found in correlation
Dmi8 inverted microscope, by Leica (3 mentions)
N n dimethyl 9 9 biacridinium dinitrate, by Merck Group (2 mentions)
Cell light edu apollo567 in vitro kit, by RiboBio (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Celltiter glo 2.0 assay kit, by Promega (1 mentions)
Ni nta agarose, by Thermo Fisher Scientific (1 mentions)
Adp glo max assay kit, by Promega (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibodies, by Affinity Biosciences (1 mentions)
Nucleoprotein extraction kit, by Sangon (1 mentions)
Caspase 3 activity assay kit, by Abcam (1 mentions)
Tunel brightgreen apoptosis detection kit, by Vazyme (1 mentions)
Mitobright lt green, by Dojindo Laboratories (1 mentions)
Celllight mitochondria rfp, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Celltox green, by Promega (1 mentions)
Cignal reporter assay kit, by Qiagen (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Rabbit anti ply antibody, by Abcam (1 mentions)
Goat anti rabbit alkaline phosphatase antibody, by Abcam (1 mentions)
25 protocols using varioskan multimode microplate reader
1
BH3 Profiling Assay for Mitochondrial Apoptosis
2
Antimicrobial Efficacy of Essential Oils
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The MIC determinations for each EO and Hd were performed by the broth microdilution method. Microbial suspensions of each strain were prepared in PBS to a final concentration of 1.5 × 108 CFU/mL for bacterial strains and 1.5 × 106 CFU/mL for the yeast and mold. Then, 100 μL Mueller Hinton (MH) for bacteria and Sabourad dextrose broth (SBD) for the yeast and mold were added to each well of a 96-well microplate. Afterwards, 100 μL of the EO and Hd solutions (1 mg/mL) prepared in the appropriate culture media were added to the first well followed by a twofold serial dilution to obtain final concentrations ranging from 500 mg/mL to 0.48 µg/mL. Finally, 10 µL of the bacterial/fungi suspension diluted in the appropriate culture medium were added to each well to obtain a final concentration of 105 CFU/mL. The microplates were incubated at 35–37 °C for 24 h for bacteria or 48 h for yeast, and at 22 °C for 5 days for Aspergillus brasiliensis. Bacterial growth and culture medium were used as controls. The minimal inhibitory concentration (MIC) was defined as the lowest concentration where no visible growth was observed, and growth was monitored by measuring OD600nm in a microplate reader (Varioskan™ multimode microplate reader, Thermo Scientific, Massachusetts, USA). All experiments were performed in triplicate.
3
Measuring Neutrophil Oxidative Burst Using Extracellular Vesicles
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PMNs (200 μl of 5 × 106/ml) were added to 2,000 μl aEV, lysed aEV, sEV, apoEV sample or HBSS at 37°C in a linear shaker (0.18 g) for 45 min. aEV, lysed aEV, and sEV samples were prepared from 4 × 107 cells, whereas apoEV samples were derived from 2 × 107 cells.
Lucigenin (5 mg/ml N,N′-dimethyl-9,9′-biacridinium dinitrate dissolved in DMSO, both from Sigma) was added in 1:100 volume ratio to the pretreated cells. White flat-bottom 96-well plates were coated with 10% FBS at room temperature for 1 h. Three parallel 180 μl samples of pretreated PMNs were activated in the coated wells with 20 μl 1 μM PMA. Changes in the luminescence were recorded for 90 min at 37°C with gentle shaking in a Varioskan multimode microplate reader (Thermo Fisher Scientific) every minute.
Lucigenin (5 mg/ml N,N′-dimethyl-9,9′-biacridinium dinitrate dissolved in DMSO, both from Sigma) was added in 1:100 volume ratio to the pretreated cells. White flat-bottom 96-well plates were coated with 10% FBS at room temperature for 1 h. Three parallel 180 μl samples of pretreated PMNs were activated in the coated wells with 20 μl 1 μM PMA. Changes in the luminescence were recorded for 90 min at 37°C with gentle shaking in a Varioskan multimode microplate reader (Thermo Fisher Scientific) every minute.
4
Quantifying Cell Proliferation and Viability
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Three thousand cells per well were seeded in 96-well plates and allowed to grow for indicated days. At the experimental endpoint, cells were incubated with a medium premixed with 10% CCK8 (Dojindo, Kumamoto, Japan) at 37 °C for 2 h, followed by monitoring absorbance at 450 nm. EdU assays were performed with a Cell-Light EdU Apollo567 In Vitro Kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. The EdU incorporation rate, as captured by a DMi8 inverted microscope (Leica), reflected cell proliferation ability. Alternatively, SRB assays were used to determine cell proliferation: In a 96-well plate, seeded cells were fixed with 10% trichloroacetic acid overnight at 4 °C and stained with 0.4% SRB for 20 min at room temperature. After washing the plates with 1% acetic acid and solubilizing the residual dye with 10 mM Tris base solution for 15 min, the absorbance was monitored at 510 nm. We performed cell viability assays using the CellTiter-Glo 2.0 Assay Kit (Promega, Madison, WI, USA) and normalized the results by the cellular protein contents measured in parallel. The absorbance and chemiluminescence intensity were quantified using a Varioskan Multimode Microplate Reader (Thermo Fisher Scientific). Experiments were repeated independently three times.
5
Purification and ATPase Assay of RUVBL1/2
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His-Flag–tagged RUVBL1 and RUVBL2 were expressed in Rosetta (DE3) pLysS competent cells and purified using Ni-NTA agarose (Life Technologies). Expression vectors were provided by M. Grigoriev (Washington University School of Medicine). ATPase assay was performed using an ADP-Glo Max Assay kit (Promega). Proteins were incubated with the compound for 10 min and added to the ATPase buffer to a final concentration of 50 mM tris-HCl (pH 7.5), 10 mM MgCl2, 20 mM NaCl, 1 mM ATP, and BSA (0.05 mg/ml). Reagents included in the kit were subsequently applied as shown in the manufacturer’s instruction to detect the adenosine 5′-diphosphate formed in the assay. The luminescence was measured using a Varioskan multimode microplate reader (Thermo Fisher Scientific).
6
Quantitative Protein Analysis Pipeline
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Total protein was obtained by RIPA buffer (New Cell & Molecular Biotech, Suzhou, China) and nuclear protein was extracted using the Nucleoprotein Extraction Kit (Sangon Biotech, Shanghai, China). Blots were incubated overnight at 4 °C with respective primary antibodies listed in Supplementary Table 2 , followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Affinity Biosciences, OH, USA) for 1 h at room temperature, then developed with high-sensitivity ECL (Vazyme, Nanjing, China). The protein bands were visualized using a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). ACTB or histone H3 were used as loading controls. The validation information of antibodies is provided in the Reporting Summary. ELISA assays of human TGFβ1 and mouse IL1β/IL6/TNFα/IL17A (Multi Sciences, Hangzhou, China) were performed as per the manufacturer’s protocol using a Varioskan Multimode Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA).
7
Caspase3 Apoptosis Assay Protocol
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The cells were exposed to either vehicles or apoptosis inducers (1:1,000, Beyotime, Shanghai, China). The activity of Caspase3 was then probed by employing the Caspase3 Activity Assay Kit (Abcam, Cambridge, UK) on a Varioskan Multimode Microplate Reader (Thermo Fisher Scientific) after 6 h, and immunoblotting was conducted to gauge the levels of cleaved Caspase3 and cleaved PARP. Subsequently, the percentage of apoptotic cells was quantified by applying the TUNEL BrightGreen Apoptosis Detection Kit (Vazyme) on a DMi8 inverted microscope (Leica) after 12 h.
8
Mitochondrial Dynamics in Co-cultured Cells
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Mitochondria in SW480 and HCT116 cells were labeled using CellLight Mitochondria-RFP (Thermo Fisher Scientific) or MitoBright LT Green (Dojindo) as per the manufacturer’s protocol. FHC cells were then co-cultured with the labeled CC cells through the Transwell system for four days, followed by the detection of fluorescence signals in each cell line with a DMi8 inverted microscope (Leica). In parallel, FHC cells educated with EVs for the indicated time periods were harvested for assessment of mitochondrial mass using MitoTracker Green FM (Invitrogen) on a Varioskan Multimode Microplate Reader (Thermo Fisher Scientific).
9
Cytotoxicity Screening of Small-Molecule Library
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SW480 shControl or SW480 shTSC2 cells were seeded to 96-well plates in DMEM with 10% FBS and penicillin/streptomycin. After overnight culturing, transfer of small-molecule library was performed. At 36-hour after compound addition, 50 μl of CellTox Green (Promega) was added to each well. The plates were shaken and allowed to sit for 10 min before being read on the Varioskan multimode microplate reader (Thermo Fisher Scientific).
10
Measuring Neutrophil Oxidative Burst Using Extracellular Vesicles
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