Caco-2 human epithelial colorectal adenocarcinoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with Gibco® 10% fetal bovine serum (FBS; Thermo Fisher Scientific). LDH-DCA-HA and INS@LDH-DCA-HA were each incubated with Caco-2 cells for 24 h. Thereafter, 10 μL MTT reagent (Beyotime, Shanghai, China) was added to the medium, according to the manufacturer’s instructions, and the optical density (OD) was measured using an enzyme-labeled instrument (TECAN, Geneva, Switzerland).
Enzyme labeled instrument
Manufactured by Tecan
Sourced in Switzerland
The Enzyme-labeled instrument is a laboratory device that utilizes enzyme-based detection methods to perform various analytical tasks. It is designed to accurately measure and quantify the presence and/or activity of specific enzymes in samples. The core function of this instrument is to facilitate enzyme-based assays and analyses, providing researchers and scientists with a reliable tool for their laboratory investigations.
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4 protocols using enzyme labeled instrument
1
Caco-2 Cytotoxicity Assay with LDH-DCA-HA
2
Quantitative ELISA-Based Cytokine Assay
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The human IFN-gamma ELISA kit, IL-2 ELISA kit, and TNF-alpha ELISA kit (all kits, Dakewe, Beijing, China) were used to measure the concentrations of IFN-γ and IL-2, TNF-α, respectively. According to the instructions of the ELISA kit, three samples were processed, and the standard curve was prepared. Then the fluorescence value was measured by the enzyme-labeled instrument (TECAN, Mannedorf, Switzerland). The cytokines quantity was then calculated.
3
Cytotoxicity Evaluation of Nanoprobes
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A cell counting kit-8 (CCK8) assay was used for cell cytotoxicity analysis. After incubation with ox-Low Density Lipoprotein (ox-LDL) for 24 h, 1 × 104 HUVECs were seeded in 96-well plates under different concentrations of nanoprobes (ICG 0, 2, 4, 8, 16, 32, 64 mg/mL). After 48 h of incubation, the relative cell viability was assessed by CCK-8 assay. CCK8 solution (10 μL) was added to each well and incubated for 2 h. The absorbance of each well at 450 nm was determined by an enzyme-labeled instrument (Tecan, Switzerland). The mean absorbance of the probe group was compared with that of the control group to obtain the cell survival ratio.
4
Cytotoxicity Assessment of XFBD in RAW264.7 and NIH-3T3 Cells
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The RAW264.7 cells were seeded into 96-well plates at a density of 4 × 104 cells per well at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. After inoculation, the cells are then treated with the different concentrations of XFBD (0, 10, 25, 50, 100, 200 μg/mL) for 24 h. NIH-3T3 cells were seeded into 96-well plates at a density of 4 × 103 cells per well. After 24 h, cells were incubated for 48 h in the presence of different concentrations of XFBD (0, 1, 2.5, 5, 10, 25, 50 μg/mL).
Cell viability was detected using MTT (methylthiazole diphenyl-tamazole). After discarding the supernatant, adding MTT (10%) and incubated 4 h. The MTT solution was removed, then the precipitation dissolved in the DMSO solution. Measure the absorbance value at 490 nm using the enzyme-labeled instrument (Tecan, Austria).
Cell viability was detected using MTT (methylthiazole diphenyl-tamazole). After discarding the supernatant, adding MTT (10%) and incubated 4 h. The MTT solution was removed, then the precipitation dissolved in the DMSO solution. Measure the absorbance value at 490 nm using the enzyme-labeled instrument (Tecan, Austria).
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