Murine fibrosarcoma MCA205 cells were treated with 2.8 µM pz I-PDT or 2.4 µM pz III-PDT (IC90) as described above. After the indicated durations, the supernatant was collected, cleared from dying tumor cells by centrifugation, and frozen at − 20 °C until HMGB1 was quantified later with an ELISA kit (IBL-Hamburg). All assays were performed in accordance with the manufacturer’s instructions. HMGB1 was quantified by using Tecan Spark 20 M microplate multimode reader. A four-parameter logistic curve fit was used for data analysis.
Spark 20 m microplate multimode reader
Manufactured by Tecan
The Spark 20 M is a microplate multimode reader manufactured by Tecan. It is designed to perform various measurements on samples in microplates. The device can detect and quantify different types of signals, including absorbance, fluorescence, and luminescence.
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3 protocols using spark 20 m microplate multimode reader
1
Quantifying HMGB1 Release in Murine Fibrosarcoma
2
Quantifying Extracellular ATP in Cell Lines
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GL261 and MCA205 cells were treated with PS-PDT or PD-PDT as described above and incubated for 24 h in a medium with 2% FBS. Then supernatants were collected and centrifuged at 15,000 rpm at 4 °C for 3 min. The supernatants were either stored at − 80 °C or used immediately for ATP measurements. ATP analysis was done by using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, G7571) as described by the manufacturer. The luminescence was measured on a Tecan Spark® 20 M microplate multimode reader.
3
Measuring Murine Fibrosarcoma ATP Levels
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Murine fibrosarcoma MCA205 cells were incubated for 24 h with 2.8 µM pz I or 2.4 µM pz III (IC85) in RPMI medium supplemented with 2% FBS as described above. The supernatants were collected and centrifuged at 15,000 rpm at 4 °C for 3 min and then stored at -80 °C for ATP measurement if that was not done immediately. ATP analysis was done according to the manufacturer’s instructions by using CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, G7571). Tecan Spark 20 M microplate multimode reader was used to quantify the luminescence.
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