Cell viability was assessed using the CellTiter-Blue Cell Viability Assay (Promega). Briefly, 1 × 105 BMDMs per well were seeded on a 96-well plate overnight. The next day, medium was replaced with fresh medium containing different concentrations of itaconate, mesaconate, DMI or 4-OI for 4 h, followed by stimulation with LPS (10 ng ml−1) overnight. Afterwards, supernatants were removed and 1× CellTiter-Blue reagent in medium was added for 40–60 min at 37 °C and then fluorescence (560Ex/590Em) was measured using the SpectraMax i3x Multi-Mode Detection System (Molecular Devices).
Spectramax i3x multi mode detection system
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax i3x Multi-Mode Detection System is a versatile lab equipment that can perform various detection methods, including absorbance, fluorescence, and luminescence. It is designed to provide reliable and accurate results for a wide range of applications in life science research.
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7 protocols using spectramax i3x multi mode detection system
1
Assessing Cell Viability in BMDMs
2
Quantification of Inflammatory Markers in Cell Culture
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Conditioned media were collected and stored at −80 °C immediately after collection. The levels of inflammatory markers, including IL8 (DY208), IL1β (DY201), TNFα (DY210), and IL6 (DY206), were measured by ELISA (R&D Systems). Conditioned media from co-cultures were centrifuged to pellet the remaining bacteria. ELISA components were reconstituted following the manufacturer’s certificate of analysis and a BD Falcon Microtest 96-well ELISA plate (R&D Systems DY990, Minneapolis, MN, USA) was coated with the corresponding capture antibody (100 µL per well) and incubated overnight at room temperature according to the manufacturer’s guidelines. Standards and samples were loaded into 96-well plate (100 µL per well) and incubated at room temperature for 2 h before detection with horse radish peroxidase-conjugated antibody (1:40; 100 µL per well) and substrates A:B (R&D Systems DY999, Minneapolis, MN, USA). Reaction was stopped with 2N H2SO4. Using the Spectramax i3x Multi-Mode Detection system (Molecular Devices, San Jose, CA, USA), and the absorbance was measured at OD 450 nm, with a wavelength correction set at 540 nm. Standard curves were plotted, and sample values were interpolated using a four-parameter logistic curve fit (second order polynomial quadratic curve) on GraphPad Prism (Prism 8).
3
E-Cigarette Aerosol Cytotoxicity Evaluation
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Epithelial cells (2.0 × 103 cells/mL seeding density in a 96-well plate; 100 µL per well) were treated with e-cigarette aerosol pretreated media for 30 min or continuously. The media was pretreated with 3, 6, and 15 puffs or nicotine free and nicotine e-cig aerosol. Treated cells were incubated with cell titer-blue reagent provided in the cell viability assay (10 µL per well; Promega G8081, Madison, WI, USA) for 1.5 h. Fluorescence is recorded at 560Ex/590Em using a Spectramax i3x Multi-Mode Detection system (Molecular Devices, San Jose, CA, USA) according to the manufacturer’s protocol. The manufacturer notes a linear relationship between cell number and fluorescence.
4
Biofilm Quantification in e-Cigarette Exposure
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Biofilm formation was measured following a modified protocol previously described [33 (link)]. TSB was pretreated with e-cigarette aerosols (6 puffs), overnight cultures were diluted 1:100 into pretreated media and incubated for 24 h in a 96-well plate (100 µL per well). The wells were washed with distilled water to remove planktonic bacteria and incubated at 37 °C to air dry for 20 min. The biofilm was incubated in 125 µL of safranin (Millipore Sigma 65092B-95, Darmstadt, Germany) at room temperature for 20 min. Safranin was discarded, and the wells were washed with distilled water, and air dried at 37 °C. The biofilm was fixed and dissociated by resuspending in a mix of ethanol and acetone (80:20) and absorbance measured at OD 490 nm using the Spectramax i3x Multi-Mode Detection system (Molecular Devices, San Jose, CA, USA). We set the OD for the control to 1 to allow for a direct comparison of the effect of treatments.
5
Measuring Bacterial Attachment to Epithelial Cells
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Changes in bacterial attachment, which is the first step during bacterial invasion, was measured following a modified protocol previously described [32 (link)]. OKF6 cells were seeded at 2.5 × 105 cells/mL in 24-well plates and incubated with e-cig pretreated S. aureus for 3 h to allow attachment to epithelial cells. The supernatant was collected, remaining attached cells were washed twice with 1×PBS to eliminate any non-attached bacteria, and 1×PBS was also collected. Then, the epithelial cells with possible internalized bacteria and attached bacteria were permeabilized by incubation in 1% Triton X-100 (100 µL for 10 min), the Triton X-100 was then diluted by adding 900 uL of sterile 1×PBS. All the collected bacteria were centrifuged (13,000× g for 5 min). Bacteria were then resuspended in 1 mL of 1×PBS and incubated with BacLight Green Bacteria stain (100 µL per well; Invitrogen B35000, Eugene, OR, USA) (1:1000 dilution) for 15 min. Bacteria luminescence values were normalized to luminescence of uninfected permeabilized epithelial cells. After incubation, bacteria were loaded into a 96-well plate, and luminescence was measured with the Spectramax i3x Multi-Mode Detection system (Molecular Devices, San Jose, CA, USA).
6
E-Cig Aerosol Impact on Bacterial Viability
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To observe the viability changes caused by e-cig vape during exponential phase and stationary phase, bacterial overnight cultures were diluted (1:10) into e-cig aerosol pretreated media (6 puffs), loaded into a 96-well plate (100 uL per well), and allowed to grow exponentially. Following the manufacturer protocol, viability was measured every hour with BacTiter-Glo Microbial Cell Viability Assay (100 uL per well; Promega G8232, Madison, WI, USA), which measures viability based on quantitation of the ATP present in the bacterial cells. The reagent was added to bacterial cells, and luminescence was measured using the Spectramax i3x Multi-Mode Detection system (Molecular Devices, San Jose, CA, USA).
7
Cell Viability and Proliferation Assay
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To determine cell viability and proliferation, 2000 cells/well were seeded in a 96 well plate. CellTiter-Blue® reagent (Promega, Madison, WI, USA) was added to the wells and incubated for an hour prior to recording fluorescence at 560/590 nm using a Spectramax i3x Multi-Mode Detection system (Molecular Devices, San Jose, CA, USA) following the manufacturer’s instructions.
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