Adenosine assay kit

Hippocampus tissue was homogenized in PBS and centrifuged at 10,000× g for 10 min at 4 °C. Next, the supernatants were collected and assayed without dilution via the Adenosine Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA, MET-5090) according to the manufacturer’s instructions. Glutamate was analyzed by a colorimetric enzyme method using a kit (Sigma Aldrich, St. Louis, MO, USA, MAK004), following the company’s instruction sheet, and presented as μmol/mL.

The levels of glycogen, glucose, and adenosine were determined in S. litura hemolymph using colorimetric methods with a glycogen assay kit (MET-5022, Cell Biolabs Inc.), glucose assay kit (Cell Biolabs Inc.), and adenosine assay kit (Cell Biolabs Inc.), respectively. Detailed procedures have been described previously⁶⁰ .

Duplicates of GL261 WT cells, GL261-CD73 overexpressing cells, or only medium (DMEM with 10% FBS and 1% penicillin/streptomycin) were seeded into 6-well plates (100,000 cells/ well) and cultured in the presence of 3 μM adenosine 5′-monophosphate (5′AMP; MilliporeSigma). After 72 hours, the supernatant was harvested, and the adenosine concentration was determined with an Adenosine Assay Kit (Adenosine Assay Kit, Cell Biolabs) according to the manufacturer’s instructions. The assay was measured with the CLARIOstar Plus plate reader (BMG Labtech).

Levels of adenosine in the mouse sera were measured using an Adenosine Assay Kit (Cell Biolabs, USA). A reaction mixture was prepared by mixing fluorometric probe (1:100), avidin-horseradish peroxidase (1:500), adenosine deaminase (1:500), purine nucleoside phosphorylase (1:10), and xanthine oxidase 1:50 in assay buffer. A control mixture containing the same contents as for the reaction mixture except for adenosine deaminase was prepared also. Adenosine standard solutions and mouse samples (50 μl) were added individually into the wells of a black microtiter plate; then either 50 μl of the reaction or a control mixture was added in duplicate. The plate was incubated (37°C, 15 min) in darkness. The excitation (530–570 nm) and emission (590–600 nm) of each well’s content was determined using a fluorescence microplate reader (BioTekTM SynergyTM H1 Hybrid Multi-Mode Monochromater Fluorescence Microplate Reader). The net relative fluorescence unit (RFU) of each sample (difference between the RFU of the wells added with reaction-mixture and the well added with control mixture of the same sample) was used to extrapolate the adenosine level in the mouse serum from the standard adenosine RFU curve.
The levels of mouse lung IDO1 were measured as described previously (19 (link)) using IDO1-ELISA kit (LifeSpan Biosciences, USA).

Hematoxylin & eosin, periodic acid–Schiff (PAS) and Masson’s trichrome (TRI) dyes were from Merck Millipore, USA. Alum adjuvant (Pierce), RevertAid H Minus Reverse Transcriptase, and Rapid DNA Ligation Kit were from Thermo Fisher Scientific, USA. Limulus amoebocyte lysate assay kit was from Biolasco, Taiwan. Adenosine assay kit was from Cell Biolabs, USA. Tetanus toxoid (TT) was from Biofarma, Indonesia. Anti-CD16/32 (FcX), anti-CD3-FITC, anti-CD4-PerCP and anti-FoxP3-Alexa Flour^®647 were from BioLegend, USA. Anti-CD25-BV421 and anti-CD45RA-PE were from BD Biosciences, USA. IDO1 ELISA kit was from LifeSpan Biosciences, USA. Phosphatidylcholine was from Lipoid-AG, Switzerland. Cholesterol was from Sigma-Aldrich, Germany. Dimethyldioctadecyl-ammoniumbromide (DDAB) was from Honeywell Research Chemicals, USA.

The production of adenosine was measured directly in the supernatants obtained during CD8+ T cell activation. For this, naive CD8+ T cells were cultured in 96-well plates at 10⁵ cells/well in the presence of soluble α-CD3 (1 μg/mL) and α -CD28 antibodies (1 μg/mL) and IL-2 (10 ng/mL). The culture medium was harvested on days 2 and 4 of activation, then centrifuged at 1000 × g for 10 min to discard cells and stored at –20°C for further analysis.
To evaluate adenosine production by Tc1 cells in the presence of AMP, Tc1 cells were differentiated for 3 days, harvested and diluted in Hanks’ balanced salt solution (HBSS). Cells were then incubated in 96-well flat-bottom plates at 0.5 × 10⁵ cells/well with AMP (10 μM, Sigma-Aldrich) in the presence or absence of the CD73 inhibitor APCP [Adenosine 5′-(a,b-methylene) diphosphate] (50 μM, Sigma-Aldrich). After 1 h, the cells were harvested, placed on ice for 15 min, and then centrifuged at 1000 × g for 10 min. Supernatants were collected and stored at –20°C until further analysis. Adenosine production was measured using the Adenosine Assay Kit (Cell Biolabs Inc.) following the manufacturer’s instructions.