To compare the remaining cell number in the cell culture after transfection with polyplexes at various N/P ratios and DNA amount, HEK293T cells were fixed by incubation in 4% paraformaldehyde for 20 min. After which, the cells were stained with 1 µg/ml of Hoechst stain 33342 in 1× PBS for 5 min. Cells were washed once with 1× PBS and subjected to RFU measurement with the Synergy H1 Multi-Mode Reader. The RFU was obtained with predetermined setting of Ex/Em at 485/585 nm and gain at 100, capturing RFU from nine positions in each well. The percentage of viable cells was quantified through the Nucleocounter. The sample preparation has been described in section on transfection efficiency analysis. The software provides the information on the number of PI− cells/0.8 µl of samples. This enables the calculations of the % of viable cells.
Synergy h1 multi mode reader
Manufactured by Synergy Software
Sourced in United States
The Synergy H1 Multi-Mode Reader is a versatile laboratory instrument designed to perform a variety of detection methods. It is capable of measuring absorbance, fluorescence, and luminescence in microplates and cuvettes.
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Lab products found in correlation
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt assay, by Merck Group (1 mentions)
Cck 8, by Synergy Software (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
White walled clear bottom 96 well plate, by Corning (1 mentions)
Nadp nadph glo, by Promega (1 mentions)
Gsh gssg glo assay, by Promega (1 mentions)
7 protocols using synergy h1 multi mode reader
1
Viability Assay for Polyplexes
2
Measurement of Cellular Reactive Oxygen Species
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Cellular reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) [45 (link),46 (link)]. Cells were plated into black-walled clear bottom 96-well plates (Corning). For time-course ROS assays, cells grown in 2 mM glutamine were seeded, and the next day, the media was changed to 4 mM glutamine for two, six, twelve, or twenty-four hours. Following treatment, media was removed, and cells were washed once with PBS. Cells were incubated in the dark at 37 °C in 10 μM DCFH-DA in PBS for 20 min. Fluorescence was measured using a Synergy H1 Multi-Mode reader (excitation/emission 485/530 nm). Fluorescence measures were normalized to cell viability, as measured by MTT.
3
Measuring Intracellular Oxidative Stress
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General intracellular oxidative stress was measured as described previously [25] . Briefly, SH-SY5Y cells were plated on black coated 24-well plates (1.9 cm2) at a concentration of 8 × 104 cells per well. After differentiation, each well was pre-treated with indicated compounds for 24 h, followed by an additional 24-h co-treatment with either AGEs or BSA control. At the end of 48 h, media was gently removed and 200 µL of 20 µM 2′,7′-dichlorofluorescin diacetate (DCFH-DA) in PBS was added to each well. Fluorescence was measured after a 20-min incubation at 37 °C and 5% CO2 using a Synergy H1 Multi-Mode Reader (ex/em. 485/530). Fluorescence units were further normalized to viable cell number using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO). Though this method has been used to measure general oxidative stress [26] (link), significant limitations exist. The method does not quantify specific oxidant species. The intermediate DCF semiquinone free radical, formed via one-electron oxidation of DCFH, reacts with molecular oxygen to form superoxide. This can result in an artifactual amplification of fluorescence intensity [27] (link), [28] (link).
4
Quantifying Covalent Bonding in MA-NG-GelMA Hydrogels
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To prove the covalent bonding of MA-NG within the GelMA network, Amine-NG was incorporated into the GelMA hydrogel as a comparison, by the same method as the MA-NG-GelMA hydrogel preparation described in Section 2.9 . As shown in Scheme 1 , after UV cross-linking, both the Amine-NG-GelMA hydrogel and MA-NG-GelMA hydrogel were crushed and vortexed vigorously in water, followed by centrifugation to spin down the hydrogel fragments. Afterward, the fluorescence intensity of the supernatant was measured at 25 °C using a Synergy H1 Multi-Mode Reader at an excitation wavelength of 548 nm and an emission wavelength of 580 nm. Ultrapure water was measured as a reference.
5
CCK-8 Assay for 3T3-L1 Proliferation
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Cell proliferation was determined by CCK-8 (Dojindo, Osaka, Japan). The 3T3-L1 preadipocytes were seeded at 1 × 103 per well in a 96-well plate and cultured in DMEM. When cells were attached to the bottom (about 6 h), different concentrations of APS (0, 0.1, 0.5, 2.5, 10 μg/mL) were added to the medium. After 24 h, 10 μL of CCK-8 reagent was added to the medium, and cells were incubated for 2 h. Finally, the absorbance at 450 nm was measured using a Microplate Reader (Synergy H1 Multi-Mode Reader, Winooski, VT, USA).
6
ATPase Activity Determination Protocol
7
Measuring Cellular Redox Ratios
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Cells were seeded into the white-walled clear bottom 96-well plates (Corning). On day two, ratios of NADPH/NADP+ and GSH/GSSG were measured with NADP+/NADPH-Glo and GSH/GSSG-Glo Assays (Promega) according to the manufacturer’s instructions. Luminescence was measured using a Synergy H1 Multi-Mode reader.
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