Before ELISA, thawed culture medium were centrifuged at 3,000 rpm for 5 min at 4°C to remove cell debris. The levels of IL-6, TNF-α, IL-1β and IFN-γ in the culture medium supernatant were assessed using Human IL-6 Quantikine ELISA Kit, Human TNF-alpha Quantikine HS ELISA, Human IL-1 beta/IL-1F2 Quantikine ELISA Kit, and Human IFN-gamma Quantikine ELISA Kit (R&D) following the manufacturer’s instructions. Absorbance at 450 nm was measured using the iMark microplate Absorbance Spectrophotometer (Bio-Rad, Philadelphia, PA, USA).
Imark microplate absorbance spectrophotometer
Manufactured by Bio-Rad
Sourced in United States, Canada
The IMark microplate Absorbance Spectrophotometer is a laboratory instrument designed for the measurement of absorbance in microplate samples. It is capable of performing absorbance measurements across a wide range of wavelengths to support various analytical applications.
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Lab products found in correlation
Human ifn gamma quantikine elisa kit, by R&D Systems (1 mentions)
Human tnf alpha quantikine hs elisa, by R&D Systems (1 mentions)
Human il 6 quantikine elisa kit, by R&D Systems (1 mentions)
Human il 1 beta il 1f2 quantikine elisa kit, by R&D Systems (1 mentions)
Biotin labeling kit nh2, by Dojindo Laboratories (1 mentions)
Ps capture exosome elisa kit, by Fujifilm (1 mentions)
3 protocols using imark microplate absorbance spectrophotometer
1
Cytokine Quantification in Cell Cultures
2
Exosome Biomarker Profiling in Urine
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Before ELISA, thawed urine samples were centrifuged at 1,200 × g for 20minat 4°C to remove cell debris and urine salts. ELISA was performed using the PS Capture Exosome ELISA Kit (Cat# 298-80601 FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) according to the manufacturer’s instructions. Absorbance at 450 nm was measured using the iMark microplate Absorbance Spectrophotometer (Bio-Rad). Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK). For the latter three antibodies, biotin was conjugated to them using the biotin labeling kit-NH2 (Dojindo, Kumamoto, Japan), according to the manufacturer’s instructions.
3
Cellular Cytotoxicity Assessment by LDH Assay
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As described in one of our previous studies [61 (link)], the cellular cytotoxicity test was performed by LDH assay using the LDH Cytotoxicity Detection Kit. Briefly, a specimen containing 2 × 105 cells was seeded in 24-well plates for 24 h. A 50 µL amount of duplicate LDH mixture solution was added to an equivalent volume from each supernatant and was prepared in a 96-well plate and then incubated for 20 min at room temperature in the dark until the yellow color developed before reading at 490 nm with an iMark microplate absorbance spectrophotometer (Bio-Rad, Mississauga, ON, Canada). The cell toxicity was represented by percentage. Treated with 1% of Triton X-100, 2 × 105 cells were used as a positive control for LDH and corresponded to 100% of cytotoxicity. This experience was repeated three times.
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