NAD+/NADH levels were measured with Amplite Fluorimetric NAD/NADH ratio assay kits (ATT Bioquest, CA) as previously described (Li et al., 2015 (link)). Briefly, ~20 mg human placental or mouse placental tissue was weighed, washed with cold PBS, and homogenized with 400 μl of lysis buffer in a microcentrifuge tube. After centrifugation at 1,000 g for 5−10 min, the supernatant was collected and used for subsequent NAD+/NADH assays. For the measurement of intracellular NAD+/NADH levels, 25 μl of cell lysate was treated with or without NAD+/NADH extraction solution for 15 min, neutralized with extraction solutions at room temperature, and incubated at room temperature in the dark for 30 min after the addition of 75 μl of NADH reaction mixture. The readings were taken by running a 96‐well black plate on a Varioskan LUX fluorescence microplate reader (Thermo Fisher Scientific, Waltham, USA) at Ex/Em = 530–570/590–600 nm (maximum Ex/Em = 540/590 nm). The blank signal was subtracted from the values for those wells with the NADH reactions.
Varioskan lux fluorescence microplate reader
Manufactured by Thermo Fisher Scientific
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The Varioskan LUX is a fluorescence microplate reader designed for a wide range of applications in life science research. It measures fluorescence intensity, time-resolved fluorescence, and luminescence in microplates. The instrument provides accurate and reliable data for various fluorescence-based assays.
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5 protocols using varioskan lux fluorescence microplate reader
1
Quantifying NAD+/NADH Levels in Tissues
2
Inhibition of AGEs Formation in BSA-Glucose System
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The non-enzymatic glycosylation system of bovine serum albumin (BSA)-glucose was constructed based on the methods reported by Amamou et al. [26 (link)]. Accurately, 10 mL of bovine serum albumin (BSA, 20 mg/mL), 5 mL of glucose solution (500 mmol/L), and 5 mL of phosphate buffer (200 mmol/L, pH 7.4) containing 0.02% (w/w) of sodium azide were mixed. This mixture was considered a negative control group and then mixed with 5 mL of different CHP solutions (0.5, 1.0, 1.5 mg/mL) to obtain the reaction system. The same treatment of aminoguanidine (AG) was considered the positive control group. After incubation for 0, 7, 14, 21, and 28 days at 37 ± 0.5 °C, 0.5 mL was taken out of the above reaction solution and diluted to 10 mL with phosphate buffer (200 mmol/L, pH 7.4), respectively. The fluorescence intensities of AGEs were detected by the Varioskan LUX fluorescence microplate reader (Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA). The excitation wavelength was set at 370 nm, and the emission wavelength was set at 440 nm. The inhibition rate of AGEs was calculated using the following Formula (6):
where A0 and A1 represent the absorbance of the negative control sample and the positive control sample or CHP sample, respectively.
where A0 and A1 represent the absorbance of the negative control sample and the positive control sample or CHP sample, respectively.
3
Screening High-Fluorescence Sirohydrochlorin Strains
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The fermentation broth of the constructed strain was used as a sample to detect the intracellular heme concentration and the fluorescence intensity of sirohydrochlorin. The fluorescence intensity of sirohydrochlorin was measured at the excitation wavelength of 375 nm and the emission wavelength of 670 nm by a Varioskan LUX fluorescence microplate reader (Thermo Fisher Scientific, CA, USA). In order to screen the high fluorescence intensity strains, the Golden Gate multi-fragment plasmid library was electrically transferred into SH0 strain. Then, the mixed bacterial system was screened in PerCP-Cy5-5-5-A (488 nm, 695/40 nm) channel by Flow Cytometer (BD Aria III, USA), and single cell with high fluorescence intensity were obtained for plate culture.
4
ACE and ACE2 Activity Assays
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ACE and ACE2 activities were measured using the ACE activity assay kit and SensoLyte390 ACE2 activity assay kit (Anaspec, Fremont, CA, United States), respectively, according to the manufacturer’s instructions with some modifications. Briefly, rhACE solution dissolved in deionized water was used for analysis. ACE activity was measured in a reaction system with 50 μl of the sample and 50 μl of the ACE substrate solution with or without BAC (10–4 to 10–2 μM). ACE2 activity was assayed in the same manner as ACE activity. All assays were performed every 10 s for 30 min at 37°C using a Varioskan LUX fluorescence microplate reader (Thermo Fisher Scientific, Waltham, MA, United States). The autofluorescence value in each assay was subtracted from the measured values to generate the final results. The relative fluorescence unit of each sample was normalized to the corresponding total protein concentration, which was measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific).
5
Synthesis and Functionalization of Fluorescent SNPs
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The SNPs synthesis protocol was given in our previous study.
[14] Briefly, the 50 mg MCM‐41 type of SNPs was stirred with 5 % one mM of acetic acid for one hour at room temperature (RT). Then, the APTES solution was added to provide aminated SNPs with a molar ratio of 0.2 and incubated on a shaker at RT overnight. The next morning, the solution was washed with 1X PBS (0.01 M phosphate‐buffered saline; NaCl‐0.138 M; KCl‐0.0027 M; pH 7.4) and centrifuged at 14,000 RPM for 5 minutes. This step was repeated three times. The 5 μg/μL amino‐modified nanoparticles were prepared in 1X PBS buffer (pH: 7.4) and loaded with 100 μM fluorescein sodium salt and rhodamine B and kept in a shaker overnight. Lastly, one μM CoV‐2 probe, shown in Table1 (E, NSP12, or NSP9), was immobilized to the fluorescein‐loaded SNPs in the PBS buffer while control sequences were immobilized to the rhodamine B‐loaded silica nanoparticles with overnight incubation for the control line as a last step of probe‐gated SNPs. The particles were finally washed thoroughly with 1X PBS buffer three times and stored at +4 °C until their use. The fluorescein and rhodamine B entrapments were calculated from the calculation of unloaded amount Thermo Scientific™ Varioskan™ LUX‐ Fluorescence Microplate Reader (λ Ex/Em.: 460/520 nm, λ Ex/Em.: 535/600 nm respectively).
[14] Briefly, the 50 mg MCM‐41 type of SNPs was stirred with 5 % one mM of acetic acid for one hour at room temperature (RT). Then, the APTES solution was added to provide aminated SNPs with a molar ratio of 0.2 and incubated on a shaker at RT overnight. The next morning, the solution was washed with 1X PBS (0.01 M phosphate‐buffered saline; NaCl‐0.138 M; KCl‐0.0027 M; pH 7.4) and centrifuged at 14,000 RPM for 5 minutes. This step was repeated three times. The 5 μg/μL amino‐modified nanoparticles were prepared in 1X PBS buffer (pH: 7.4) and loaded with 100 μM fluorescein sodium salt and rhodamine B and kept in a shaker overnight. Lastly, one μM CoV‐2 probe, shown in Table
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