TSG-6 levels were determined as previously described [27 (link)] with some modifications. Briefly, wells of a 96-well plate (Costar) were coated for 6–8 h with 10 µg/ml of TSG-6 monoclonal antibody (clone A38.1.20; Santa Cruz Biotechnology) in 100 µl of PBS. The wells were washed 3 times with 400 µl of 1× wash buffer (R&D Systems), blocked with 100 µl of PBS containing 0.5% BSA (Thermo), then incubated on an orbital shaker (300 rpm) with 100 µl of sample (1:20 dilution) or recombinant human TSG-6 protein standards (R&D Systems) prepared in PBS containing a 1:20 dilution of non-conditioned medium (MEMα supplemented with 2% FBS, Pen-Strep, and L-glutamine). After 3 h, wells were sequentially washed, incubated for 2 h with 0.5 µg/ml biotinylated polyclonal goat anti-human TSG-6 (R&D Systems) in 100 µl of blocking solution, washed again, then incubated for 20 min with streptavidin-HRP (R&D systems). Following a final wash step, 100 µl of substrate solution (R&D Systems) was added to each well. The colorimetric reaction was terminated after 10 min with 2 N sulfuric acid (R&D systems). Optical density was determined using the plate reader.
2n sulfuric acid
Manufactured by R&D Systems
Sourced in United States
2N sulfuric acid is a laboratory reagent commonly used in various analytical and experimental procedures. It is a clear, colorless, and highly corrosive liquid solution with a concentration of 2 normal (2N) of sulfuric acid (H2SO4). The core function of 2N sulfuric acid is to provide a standardized acidic solution for titrations, pH adjustments, and other chemical reactions requiring a known concentration of sulfuric acid.
Automatically generated - may contain errors
Lab products found in correlation
Multiskan fc microplate photometer, by Thermo Fisher Scientific (2 mentions)
Cytotoxicity detection kit, by Roche (2 mentions)
Substrate solution, by R&D Systems (1 mentions)
Wash buffer, by R&D Systems (1 mentions)
Streptavidin hrp, by R&D Systems (1 mentions)
96 well plate, by Corning (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Softmax pro 6, by Molecular Devices (1 mentions)
Goat anti chicken igy h l hrp, by Abcam (1 mentions)
Anti neu5gc antibody kit, by BioLegend (1 mentions)
Spectramax 190, by Molecular Devices (1 mentions)
Streptavidin conjugated horseradish peroxidase, by R&D Systems (1 mentions)
Xmark spectrometer, by Bio-Rad (1 mentions)
Gal 3, by R&D Systems (1 mentions)
C reactive protein (crp), by R&D Systems (1 mentions)
Elisa, by R&D Systems (1 mentions)
Mmp 9, by R&D Systems (1 mentions)
Tmb color substrate, by R&D Systems (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Hrp conjugated donkey anti goat igg antibody, by Jackson ImmunoResearch (1 mentions)
7 protocols using 2n sulfuric acid
1
Enzyme-Linked Immunosorbent Assay for TSG-6 Quantification
2
Quantifying Cellular Damage via LDH Assay
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The activity of the LDH enzyme was measured as an indicator of cellular damage. The enzymatic activity of LDH was detected in culture supernatants using the “Cytotoxicity detection kit” (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, after 24 h, 48 h, and 72 h of culture, 100 µL of the supernatant was transferred in triplicate to a 96-well plate. Subsequently, 100 µL of the kit’s reaction mixture (catalyst and dye solution) was added to each well. After 30 min of incubation in darkness at room temperature, the reaction was stopped by adding 50 µL of 2N sulfuric acid (R&D Systems, Minneapolis, MN, USA), and its optical density (OD) was measured at 450 nm using a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific™). Triton X-lysed tissue was used as a positive control for the technique, representing 100% of the LDH activity.
3
Cytotoxicity Determination via LDH Release
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Lactate dehydrogenase (LDH) is an enzyme located in the cytosol, and it is released to culture medium upon cell damage or lysis. Therefore, LDH activity in culture supernatant can be used as a surrogate of cell membrane integrity and thus reflects cytotoxicity. LDH activity was measured using the commercial kit from Roche Diagnostics, "Cytotoxicity Detection Kit," following the manufacturer's recommendations. The evaluation involved a two-step reaction. At first, LDH present in the supernatant of cultures reduces NAD+ to NADH plus H+ by oxidation of lactate to pyruvate. Second, diaphorases catalyze the reduction of tetrazolium salt (INT) to formazan.
After 24 h of HPEs culture, 100 µL of the supernatant was transferred to a 96-well plate in triplicate. Then, 100 µL of the reaction mixture (catalyst and dye solution) from the kit was added to each well. The plate was incubated in darkness at room temperature for 30 min. After that, 50 µL of 2N sulfuric acid (R&D Systems) was added to stop the reaction. The optical density (OD) was measured at 450 nm using a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific™ Massachusetts, United States). Triton X-lysed tissue was used as a positive control to validate the technique, representing 100% LDH release (Additional file2 ).
After 24 h of HPEs culture, 100 µL of the supernatant was transferred to a 96-well plate in triplicate. Then, 100 µL of the reaction mixture (catalyst and dye solution) from the kit was added to each well. The plate was incubated in darkness at room temperature for 30 min. After that, 50 µL of 2N sulfuric acid (R&D Systems) was added to stop the reaction. The optical density (OD) was measured at 450 nm using a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific™ Massachusetts, United States). Triton X-lysed tissue was used as a positive control to validate the technique, representing 100% LDH release (Additional file
4
Enzymatic Detection of Neu5Gc in Samples
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According to the manufacturer’s recommendations, Neu5Gc was detected using an ELISA with an Anti-Neu5Gc antibody kit (BioLegend, San Diego, CA, USA; free of Neu5Gc). Briefly, serum (10 μl/well), conditioned medium (10 μl/well), and cell lysate (500 μg/well) samples collected under XC or XF conditions were placed on a high-binding 96-well ELISA plate (Corning). After the sodium carbonate-bicarbonate immobilization buffer (50 mM, pH 9.5, Invitrogen, Carlsbad, CA, USA) was added to each well, the samples were incubated for 16∼18 h at 4℃. The wells were blocked for 1 h at room temperature with the Neu5Gc Assay Blocking Solution and incubated for 2 h with a purified anti-Neu5Gc antibody (primary antibody) and chicken IgY isotype control diluted at a ratio of 1：5,000 in PBS TweenTM 20 buffer (PBST) (Pierce Biotechnology, Rockford, IL, USA). The plates were washed three times with PBST and subsequently incubated for 1 h at room temperature with goat anti-chicken IgY H&L (HRP) (Abcam) diluted at a 1：5,000 ratio in PBS. After washing three times with PBST, the wells were developed with a substrate reagent pack and the stop solution 2N sulfuric acid (R&D Systems, Minneapolis, MN, USA). Absorbance was measured at 490 nm using the Spectra-Max 190 with SoftMax Pro 6.3 software (Molecular Devices).
5
Quantitative Biomarker Measurement via ELISA
6
Mouse Serum/BAL C3 ELISA Protocol
7
Quantification of TWEAK and sICAM-1 in Vitreous Fluid
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The quantification of human TWEAK and sICAM-1 in the vitreous fluid was determined using ELISA kits according to the manufacturer's instruction. For each ELISA kit, the undiluted standard served as the highest standard and calibrator diluents served as the zero standard. Depending on the detection range for each ELISA kit the supernatant vitreous fluids were adequately diluted with calibrator diluents supplied with ELISA kit.
For the measurement of TWEAK and sICAM-1, 100 µL of 4-fold diluted vitreous samples were analyzed in the respective ELISA assays. As instructed in the kit manual, samples were incubated into each well of ELISA plates. Antibodies against TWEAK and sICAM-1 conjugated to horseradish peroxidase were added to each well of the ELISA plate.
After incubation, substrate mix solution was added for colour development. The reaction was stopped by the addition of 2N sulfuric acid (R & D systems) and optical density was read at 450 nm in a microplate reader. Each assay was performed in duplicate. Using the 4-parameter fit logistic (4-PL) curve equation, the actual concentration for each sample was calculated.
For the diluted vitreous fluids, the correction read from the standard curve obtained using 4-PL was multiplied by the dilution factor to calculate the actual reading for each sample.
For the measurement of TWEAK and sICAM-1, 100 µL of 4-fold diluted vitreous samples were analyzed in the respective ELISA assays. As instructed in the kit manual, samples were incubated into each well of ELISA plates. Antibodies against TWEAK and sICAM-1 conjugated to horseradish peroxidase were added to each well of the ELISA plate.
After incubation, substrate mix solution was added for colour development. The reaction was stopped by the addition of 2N sulfuric acid (R & D systems) and optical density was read at 450 nm in a microplate reader. Each assay was performed in duplicate. Using the 4-parameter fit logistic (4-PL) curve equation, the actual concentration for each sample was calculated.
For the diluted vitreous fluids, the correction read from the standard curve obtained using 4-PL was multiplied by the dilution factor to calculate the actual reading for each sample.
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