Cells from the AU-565 human breast cancer cell line were seeded in 96-well plates at a density of 6,000 cells/well with 100 μl/well of RPMI-1640 medium containing 10% fetal bovine serum (FBS). The cells were then cultured for 24 h in a humidified atmosphere with 5% CO2 at 37 °C. Subsequently, the cells were continuously cultured for 24 h in the same volume of fresh medium containing various concentrations of phospholipids (2.5–640 μg/ml). The medium was then replaced with 100 μl of fresh medium containing 10 μl of MTT solution (5 mg/ml), and the cells were incubated for 4 h. After the MTT-containing medium was discarded, 100 μl of dimethyl sulfoxide was added to each well to dissolve the substrate. After low-speed oscillation for 10 min, the absorbance of each well at 494 nm was measured using an Infinite F200 multimode plate reader (Tecan, Männedorf, Switzerland).
Infinite f200 multimode plate reader
Manufactured by Tecan
Sourced in Germany, Switzerland
The Infinite F200 Multimode Plate Reader is a versatile instrument designed for a wide range of detection methods. It offers fluorescence, luminescence, and absorbance measurements in a compact and user-friendly format.
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7 protocols using infinite f200 multimode plate reader
1
MTT Cytotoxicity Assay for AU-565 Cells
2
Cytotoxicity Assessment of Heat Stress
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A CCK-8 assay was conducted to assess the cytotoxicity of heat stress at different temperatures. Cells were seeded onto 96-well plates at a density of 2 × 103 cells per well and then incubated for 72 h. Then, the medium was replaced with 200 μL of medium containing 10 μL of CCK-8. After an additional 4 h incubation, the absorbance of formazan crystals was measured with an Infinite F200 Multimode plate reader (Tecan, Crailsheim, Germany) at 490 nm. All experiments were conducted in triplicate.
3
Cytotoxicity Evaluation of PEALCa and SPIO-PEALCa
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Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the cytotoxicity of PEALCa and SPIO-PEALCa complexes. Human (CRC) LoVo cells were cultured in MEM supplemented with 10% FBS and incubated at 37 °C in a humidified atmosphere with 5% CO2. In MTT assays, the cells were seeded in 96-well plates with the density of 5000 cells/well. Then the cells were incubated in fresh medium with various concentrations of PEALCa or SPIO-PEALCa (0–300 µg/mL) for 24 h. Then the PEALCa- or SPIO-PEALCa-contained MEM was replaced by fresh medium with MTT solution (Sigma, St Louis, MO) at 5 mg/mL and incubated for 4 h; subsequently, 100 µL dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazen after the MTT-contained medium was aspirated. Finally, the absorbance at 494 nm of each well was recorded using the Infinite F200 multimode plate reader (Tecan, Männedorf, Switzerland). Three duplicates were measured for each experimental point.
4
Cell Viability Assay Protocol
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Hep3B, SMMC-7721, and BEL7402 cells were seeded in 96-well plates at the density of 1 × 103 cells per well and incubated overnight. Next day, 50 ng of each plasmids were transfected into cells and incubate for another 24 h. Then, 150 μL of growth medium was replaced into each well and then 20 μL of MTT solution (5 mg/mL in PBS) was added at each time point. After cells were further incubated for 4 h, the supernatant in the wells was discarded and 150 μL of DMSO was added to dissolve the substrate for 10 min. After gentle agitation for 5 min, the absorbance at 570 nm in each well was recorded on a Tecan Infinite F200 Multimode plate reader.
5
Viability of Labeled hUC-MSCs
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A total of 5×103 hUC-MSCs or labeled hUC-MSCs were seeded per well with 100 µl culture medium in 96-well plates. The culture medium was carefully replaced following 1, 3, 12, 24 and 48 h of incubation at 37°C and 5% CO2 with Cy7-GD2. CCK-8 (10 µl) solution was added to each well and incubated for an additional 2 h. The absorbance at 450 nm was measured on an Infinite F200 Multimode plate reader (Tecan Deutschland GmbH, Crailsheim, Germany). Cell viability was calculated by the labeled hUC-MSCs/unlabeled hUC-MSCs absorbance ratio.
6
Cytotoxicity Evaluation of Nds-IR780
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All types of tumor cells in this research were purchased from ATCC. The CMM cells SK-MEL-28 were selected to evaluate the cytotoxicity of Nds-IR780 in vitro through a Cell Counter Kit-8 (CCK-8) assay. First, SK-MEL-28 cells were incubated in 96-well plates at a density of 5000 cells per well and cultured with MEM media in a humidified atmosphere with 5% CO2 at 37 °C. After 24 h, the culture media was substituted with the same volume of fresh MEM media containing various diluted concentrations of Nds-IR780 (an initial addition of 150 μg IR-780 iodide and dilutions of 60×, 50×, 40×, 30×, 20×, 10×, and 5×) for 24 h. Each single concentration was repeated in five wells. Next, 10 μL of the CCK-8 reagent was added to each well and incubated for 4 h. Lastly, the absorbance of each well was measured at 450 nm by an Infinite F200 multimode plate reader (Tecan, Männedorf, Switzerland). The cell survival rates at various concentrations of Nds-IR780 were calculated via the formula C = (A − A0)/(A1 − A0) ×100% (where C: the cell survival rate; A: the absorbance of experimental wells; A0: the absorbance of blank wells; and A1: the absorbance of control wells).
7
Cytotoxicity Evaluation of Nanocomplexes
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CCK8 assay was conducted to assess the cytotoxicity of the nanocomplexes. C6 cells were seeded in 96-well plates at 1×103 cells per well and then incubated for 12 h in 100 μL of RPMI-1640 containing 10% FBS. They were then incubated for 96 h in the Fa-deficient and FBS-free medium containing the following samples: 1) PEG-PEI-PCL micelle carrying SCR (PEC@SCR); 2) Fa-PEG-PEI-PCL micelle carrying SCR (FaPEC@SCR); 3) Fa-targeted micelle loading TMZ and siRNA (TMZ-FaPEC@siRNA, TMZ-FaPEC@SCR); and 4) non-Fa-targeting micelle loading TMZ and siRNA (TMZ-PEC@siRNA, TMZ-PEC@SCR). siRNA concentration was 20 nM in each well for all groups. The medium was then changed to 150 μL serum-free growth medium and 100 μL 1640 medium containing 10 μL CCK8. After an additional 4 h incubation, the absorbance of formazan crystals was measured on Infinite F200 Multimode plate reader (Tecan, Crailsheim, Germany) at 450 nm after 4 h incubation. All experiments were conducted in triplicate.
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