PASMCs (1 × 104 cells/well) were seeded in 96-well plates, covered with 100 μL of medium in each well for 24 h, and subsequently exposed to different treatments in accordance with the group assignment. The cell viability was determined by a Cell Counting Kit-8 and a microplate reader (Multiskan MK33; Thermolab Systems, Helsinki, Finland) at a wavelength of 450 nm.
Multiskan mk33
Manufactured by Thermo Fisher Scientific
Sourced in Finland
The Multiskan MK33 is a compact and versatile microplate reader designed for various laboratory applications. It features a wide measurement range, supports multiple detection modes, and offers user-friendly operation. The core function of the Multiskan MK33 is to provide accurate and reliable absorbance measurements for a wide range of microplate-based assays.
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9 protocols using multiskan mk33
1
Cell Viability Assay for PASMCs
2
Myocardial Cytokine Quantification
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Interleukin (IL)‐6, IL‐1β, and tumor necrosis factor (TNF)‐α levels in the homogenized myocardial tissue and cell supernatant were measured with ELISA kits (MLB00C, D6050, and DTA00D; R&D Systems) according to the manufacturer's instructions. Briefly, the tissue homogenate was processed by centrifugation at 1000×g for 10 min at 4°C. The supernatants were then cultured with the ELISA reagent. Finally, the optical density (OD) was measured using a microplate reader (Multiskan MK33; Thermolab Systems).
3
Evaluating H9c2 Cell Viability via CCK-8 Assay
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We assessed cell viability using a cell counting kit-8 (CCK-8, No. C0038, Beyotime Institute of Biotechnology, Haimen, China). We seeded H9c2 cells in 48-well plates for 24 h. Then, we infected the cells with lentivirus for 48 h before subjecting them to H/R treatment. Cells were provided with fresh media, and 10 μl of CCK-8 solution was added to every well. We then incubated the plates under normal conditions for 2 h. We measured optical density values at 470 nm using a microplate reader (Multiskan MK33, Thermolab systems, Helsinki, Finland). We repeated each experiment at least three times.
4
Cell Viability Quantification by CCK-8
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A cell count kit‐8 (CCK‐8, Beyotime, China) was employed in this experiment to quantitatively evaluate HaCaT viability. Briefly, approximately 1 × 104 cells were seeded on each film placed in the 24‐well plates for 24 h. Approximately 900 μl serum‐free DMEM medium and 100 μl CCK‐8 solution were added to each sample, followed by incubation at 37°C for several time. Supernatant was transferred to 96‐well plate, the optical density (OD) at 450 nm was determined using a microplate reader (Multiskan MK33, Thermo lab systems, Finland).
5
Cell Viability Quantification Using CCK-8
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A Cell Counting Kit-8 (CCK-8; Beyotime Biotech) was employed in this experiment to quantitatively evaluate cell viability. Following 24, 48 and 72 h of plasmid transfection, a CCK-8 assay was performed using the HTR-8/SVneo cells. Briefly, the HTR-8/SVneo cells were seeded at 0.5×104/well in 96-well plates. RPMI-1640 culture medium (100 µl/well) was added to 10 µl CCK-8 reagent after 20, 44 and 68 h and incubated at 37°C for a further 4 h, to form water-soluble formazan. The absorbances at 450 and 630 nm (calibrated wave) were determined using a microplate reader (Multiskan MK33; Thermo Fisher Scientific, Inc.). RPMI-1640 medium containing 10% CCK-8 was used as a control. The untreated control was set at 100%, and the treated samples were normalized to this value according to the following equation: survival rate (%)=optical density (OD) of the treated cells - OD of blank control/OD of negative control - OD of blank control x 100.
6
Cell Viability Assay with CCK-8
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The cells were seeded in a 96-well plate at a density of 2000 cells/well at 12 h after transfection. Cell Counting Kit-8 (CCK-8) was employed to assess cell viability. CCK-8 (10 μL, C0038, Beyotime Institute) and DMEM (100 μL) were added onto every pre-cultured film, followed by incubation of the plate under 37 °C for 3 h. At a wavelength of 450 nm, the absorbance was determined by a microplate reader (Multi-skanMK33, Thermo Electron Corporation, P.R.C)
7
Hemolysis Evaluation of Nanofiber Materials
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The hemolysis rate was evaluated by determining the hemoglobin concentration released by diluted red blood cells (RBC) exposed to the electrospun nanofiber material. The negative control consisted of adding 50 μL of RBC suspension (16% in 1× PBS, v/v) to a centrifuge tube in 2 mL 1× PBS (as a negative control). As a positive control, 2 mL deionized water was used in place of 1× PBS in order to induce maximum red blood cell lysis (n = 4). After incubating for a selected period of time, the RBC suspension was centrifuged at 2000 rpm for 5 min, and the supernatant was collected. Then, a microplate reader (Multiskan MK33, Thermo electron corporation, China) was used to measure the absorbance of hemoglobin (HB) released in the supernatant (200 μL) at 540 nm. By comparing the absorbance values of the tested supernatant and the positive control (i.e., 100% hemolysis), the hemolysis rate in the presence of different nanofibers was calculated.
8
Cell Viability Evaluation of Multilayer Films
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In this experiment, CCK-8 (Beyotime, Haimen, People’s Republic of China) assay was employed to quantitatively evaluate the cell viability of multilayer films toward HLECs. After inoculating for 24 hours, the HLECs were replaced by 100 µL 10% fetal bovine serum Dulbecco’s Modified Eagle’s Medium/F12 (1:1) mixed medium containing 10 µL CCK-8. The mixed medium was incubated to form water dissoluble formazan at 37°C for 2 hours. Then, 100 µL of the formazan solution was aspirated from each sample with a pipette and added to a new 96-well plate. The absorbance at 450 nm (calibrated wave) was examined using a microplate reader (Multiskan MK33; Thermo Electron Corporation, Shanghai, People’s Republic of China). Tissue culture plates (TCPS) without any modified films were used as control and six parallel replicates were prepared.
9
Quantifying Anti-Adhesive Brush Coatings
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Bovine serum albumin (BSA) adsorption test was performed to estimate the anti-adhesive effect of brush coatings using a bicinchoninic acid protein assay kit (Beyotime). Firstly, the 0.5 mg/mL BSA solution was prepared by diluting the standard solution (5.0 mg/mL) in the PBS solution. The BSA protein was incubated with the coating modified and pristine PDMS in a 96-well plate for 2 hours. Then, the loosely bound protein layer was removed by rinsing the sheets in a rectangular chamber containing distilled water for 1 minute. After washing with distilled water to remove the loosely bound protein, each well of a 96-well plate was injected with 0.2 mL bicinchoninic acid working solution. The microplate reader (Multiskan MK33; Thermo Electron Corporation) was used to measure the absorbance at 570 nm after incubation for 30 minutes at 37°C.
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