For the detection of cTnI and H-FABP, 250 µg (250 µL; 1 mg/mL) of polyclonal goat anti-human cTnI and 100 µg (100 µL; 1 mg/mL) polyclonal rabbit anti-human FABP were used as purchased in PBS buffer (cTnI: 0.1% NaN3; FABP: 0.02% NaN3, 0.1% BSA), pH 7.2. NHS-Fluorescein (Thermo Scientific) was dissolved in DMSO to a concentration of 10 mg/mL and added dropwise to the Pab solutions at room temperature (24 µL and 40 µL, respectively). This was reacted in darkness at room temperature for two hours on a shaker (Southwest Science LabMini MiniMixer). The crude reaction mixtures were added to dialysis cups (Thermo Scientific) with a molecular weight cut-off of 3,500 Daltons. The labeled protein was dialyzed in 100 mM PBS with 0.02% NaN3 and 0.1% Tween 20, pH 7.2 overnight. The dialyzed NHS-Fluorescein-Pab solutions were analyzed for absorbance measurements utilizing the same method as for anti-human myoglobin Pab.
Dialysis cups
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dialysis cups are laboratory equipment used to facilitate the dialysis process. They provide a contained environment for the dialysis of samples, allowing for the selective transfer of molecules across a semi-permeable membrane. The core function of dialysis cups is to enable the separation and purification of compounds based on their molecular size and diffusion rates.
Automatically generated - may contain errors
3 protocols using dialysis cups
1
Fluorescent Labeling of Cardiac Biomarkers
2
Cellular Uptake of Labeled CDNs
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All the samples used for cellular association and confocal microscopy analysis were labeled with Cyanine3 N-hydroxysuccinimide (Cy3-NHS) monoester as per the supplier’s recommendations. Lyophilized CDNs (S1, T1, and W) were labeled after redispersion in milli-q water, which maintained the same salt properties and pH of the PBS buffer before lyophilization. Labeled samples, which had a size of about 200 nm and a zeta potential of −7.37 mV, were dialyzed twice in dialysis cups (10 K MWCO; ThermoFisher Scientific) overnight in PBS.
For in vitro cellular uptake, 2.5 × 105 HeLa or CT26 cells per well were seeded in 6-well plates, or 1 × 106 BMDM per well were seeded in 12-well plates, incubated at 37 °C, 5% CO2. After 24 h, cells were treated with Cy3-labeled samples for 1 and 4 h. Then, cells were trypsinized and centrifuged at 450× g for 10 min. Cells were re-suspended in 500 µL PBS for analysis by BD LSR Fortessa flow cytometer at PE channel. Non-treated cells were used as control. In total, 10,000 events were analyzed, and data were processed using Flowjo software (Version 10.6, Ashland, Oregon, USA).
For in vitro cellular uptake, 2.5 × 105 HeLa or CT26 cells per well were seeded in 6-well plates, or 1 × 106 BMDM per well were seeded in 12-well plates, incubated at 37 °C, 5% CO2. After 24 h, cells were treated with Cy3-labeled samples for 1 and 4 h. Then, cells were trypsinized and centrifuged at 450× g for 10 min. Cells were re-suspended in 500 µL PBS for analysis by BD LSR Fortessa flow cytometer at PE channel. Non-treated cells were used as control. In total, 10,000 events were analyzed, and data were processed using Flowjo software (Version 10.6, Ashland, Oregon, USA).
3
MSC-Derived Nanoparticle Wound Healing
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Macroscopic photograph of wounds of mice that received MSC-CDNs were recorded daily post wounding using regular handheld mobile device before euthanasia on Day 7. The wound size was measured along the leading edge of newly generated epidermis and analysed using ImageJ software version 1.52 (National Institutes of Health, Bethesda, MD, USA). The percentage of wound closure was calculated based on Days 0 and 7 of the same wound and compared between treatment and control. The harvested wound tissues were prepared for brightfield imaging using the scanner, AxioScan.Z1 (Carl Zeiss Microscopy GmbH, Jena, Germany), by staining with haematoxylin and eosin (H&E) and picrosirius red (PSR). Mice that received Cy5.5-MSC-CDNs (MSC-CDNs were labelled with Cyanine5.5 N-hydroxysuccinimide (Cy5.5-NHS) monoester (Lumiprobe) as per supplier's recommendations and were dialysed in dialysis cups (10 kDa MWCO; ThermoFisher Scientific) overnight changing PBS twice to remove free dye (Cy5.5) were euthanised on Day 2 for fluorescence imaging using IVIS SpectrumCT (PerkinElmer Inc, Waltham, MA, USA) to identify any off-site interaction of MSC-CDNs at the saline control wound.
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