Spectramax m2 microplate reader

Where possible, the capacity of rSmCI-1 to cleave structural molecules was assessed using commercially available kits. Due to the variability of activity between buffers, we elected to use the KSRB from the Generic MMP Activity Kit for these assays. All MMPs were activated via incubation with 1mM APMA as previously described. Gelatin and collagen type 4 cleavage were examined using an EnzChek Gelatinase/Collagenase Assay Kit (Thermo Fisher Scientific), with MMP-2 (AnaSpec) and Clostridium histolyticum collagenase (Thermo Fisher Scientific) used as positive controls. Proteases were incubated with 100 μg/ml fluorescein labeled substrate in a clear-bottom black-welled 96 well plate and 495nm/515nm fluorescence readings were obtained after 20 hours incubations at 37°C using a SpectraMax M2 microplate reader (Molecular Devices). Differences between treatments were assessed via One-Way Anova.
Elastin cleavage was examined using a SensoLyte Green Elastase Assay Kit (AnaSpec). Porcine elastase (AnaSpec) and MMP-12 (R&D Systems) were used as positive controls. Cleavage of the 5-FAM/QXL 520 labeled elastin was measured in a clear-bottom black-welled 96 well plate and 490nm/520nm fluorescence readings were obtained after 1 hour at 37°C using a SpectraMax M2 microplate reader (Molecular Devices). Differences between treatments were assessed via One-Way Anova (Prism 9; GraphPad Software).

Bis-ANS is an extrinsic fluorescence probe used to assess the solvent-accessible apolar region on the surface of the protein and give fluorescence just after binding to the exposed hydrophobic region of the protein [47 (link)]. To assess the change in surface hydrophobicity, 2 μM WT NS and cysteine variants were incubated at 25°C with Bis-ANS (Sigma, St. Louis MO, U.S.A.) at a 1:5 ratio in dark [13 (link)]. An excitation wavelength of 390 nm was used to detect the Bis-ANS fluorescence intensity measurement [48 (link)]. Spectra were recorded from 400 to 650 nm on spectra Max M2 Microplate Readers (Molecular Devices, U.S.A.).

Images of bacteria expressing plasmid-borne gfp fusions and grown overnight on LB plates were taken using a CCD camera in Gel Doc XR⁺ image analyzer (Bio-Rad) under the SYBR Green mode. The aliquots of each strain were scraped from the plates and resuspended into PBS buffer. Cell densities were adjusted to OD_620nm ≈ 1.5 and 200 μL of bacterial suspensions were placed into 96-well microtiter plates. Two independent cultures as biological replicates and three aliquots as technical replicates were used throughout the study for each strain. Green fluorescence images were captured by SpectraMax M2 MicroplateReaders (Molecular Devices) with an excitation/emission wavelength of 485/525 nm. Fold changes in the MicF-mediated OmpF expression were calculated by dividing the specific fluorescence of strains with MicF overexpression by that of strains with negative control plasmid. The data were analyzed using One-Way analysis of variance (ANOVA) and Sidak's multiple comparisons test, where P-values of < 0.01 were considered significant.

Drug testing was conducted in ultralow attachment 96-well cell culture plates (Corning). Briefly, the LCAs or LCOs encapsulated in GelMA-Matrigel cultured for 3 days were changed with fresh media. Then, medium containing 10 ~ 20 LCAs (80 µL) was seeded into a 96-well ULA plate (Corning) using a multichannel pipette (Eppendorf). Next, LCA medium containing the drugs (30 µL) was added to the wells. The plates were incubated at 37 °C for 3 days, and the cell viability was measured by using CellTiter-Glo 3D reagent (Promega) according to the manufacturer’s instructions. The readout was performed by measuring the luminescence signal by using SpectraMax M2 Microplate Readers (Molecular Devices). The chemotherapy and targeted therapy drugs are listed in Supplementary Table 4.

Inhibition of tPA by WT NS, H138C, W154C-H138C (0–800 nM) and labeled H138C protein (0–400 nM) were monitored at 405 nm absorbance in the presence of 800 µM chromogenic substrate CH₃-SO₂-D-HHT-GLY-ARG-P-NITROANILIDE.AcOH (T-2943, Sigma-Aldrich, St. Louis MO, U.S.A.) [45 (link)]. NS and tPA substrate were added to the microtiter plate followed by the immediate addition of a constant amount of 19 nM tPA. The progressive curve for WT NS and cysteine variants (including labeled H138C) were observed for 120 min by the release of pNA upon hydrolysis of chromogenic substrates by the tPA [52 (link)]. Control protease activity was measured using the same method except for the sample containing no NS. All kinetic experiments were performed at 25°C in kinetic buffer [50 mM Hepes, 150 mM NaCl and 0.1% Tween-20 (pH 7.4)] on spectra Max M2 Microplate Readers (Molecular Devices, U.S.A.).

For quantitative Real-time Polymerase Chain Reaction (qRT-PCR) analysis, mRNA was extracted using TRIzol method and quantified in SpectraMax M2 Microplate Readers (Molecular Devices Inc., San Jose, CA, USA). The cDNA was synthesized using amfiRivert cDNA synthesis Platinum Master Mix (GenDEPOT, Barker, TX, USA). The qPCR was run using the LightCycler^® 480 SYBR Green I Master (Roche, Basel, Switzerland) in LightCycler^® 480 Instrument II (Roche Basel, Switzerland). The following primer pairs were used; for human 18s rRNA gene 5’-GGCGCCCCCTCGATGCTCTTAG-3’ and 5’-GCTCGGGCCTGCTTTGAACACTCT-3’; for human Filaggrin gene, 5’-GGAGTCACGTGGCAGTCCTCACA-3’ and 5’-GGTGTCTAAACCCGGATTCACC-3’; for human Involucrin gene, 5’-CCGCAAATGAAACAGCCAACTCC-3’ and 5’-GGATTCCTCATGCTGTTCCCAG-3’, for human Laminin 5 gene, 5’-GGAACTTCCGGCATACGGAGA-3’ and 5’-GGACAGGCACAGCTCCACATT-3’, for human Keratin 10 gene, 5’-CCGGAGATGGTGGCCTTCTCTCT-3’ and 5’-GGCCTGATGTGAGTTGCCATGCT-3’.

5 w/v% Gtn-Tyr solution was prepared by dissolving freeze-dried precursor into 1 × PBS. 400 pmol FAM-SPARC was then loaded into the 0.1 ml Gtn-Tyr solution. The precursor mixture was vortexed thoroughly to ensure the siRNA was distributed homogeneously inside the gel before the crosslinking process. HRP and H₂O₂ with a final concentration of 0.12 unit/ml and 3 mM respectively were added to the mixture and then cast into 96 well plates. The sample was allowed to set for 0.5 h before topping up with 0.1 ml of 1 × PBS as a release buffer solution. As no multiple steps or washing is involved during the siSPARC encapsulation, 100% siSPARC loading efficiency was expected using our synthesis and fabrication strategy. At a specific time, the release buffer solution was collected and replenished with fresh solution. The collected release buffer solution was then scanned using an excitation wavelength of 492 nm and an emission wavelength of 517 nm (SpectraMax M2 microplate readers, Molecular Devices, US). The fluorescence intensity was compared with the fluorescence intensity of a known amount of FAM-SPARC.