Deproteinizing sample preparation kit

In the cytosolic- and mitochondria-enriched fractions, and lactate and pyruvate levels were assessed using a Lactate Colorimetric/Fluorometric Assay Kit (BioVision, Milpitas, CA, USA, Cat# K607-100) and the Pyruvate Activity Colorimetric/Fluorometric Assay Kit (BioVision, Milpitas, CA, USA, Cat# K609-100), respectively. Fractions were deproteinized with the use of a Deproteinizing Sample Preparation Kit according to the manufacturer’s recommendations (BioVision, Milpitas, CA, USA, Cat# 808-200). Samples were transferred into a 96-well plate and mixed with Reaction Mix. After 30 min of incubation, the level of lactate was determined by colorimetric measurements at λ = 570 nm, while pyruvate was determined via fluorometric measurements (excitation wavelength λ = 535 nm, emission wavelength λ = 590 nm; Tecan Infinite 200 Pro plate reader, Mannedorf, Switzerland). The concentrations of the enzymes were calculated based on the standard curve and presented as ng/mg of protein. Protein level was measured before deproteinization.

The presence of various enzymes and proteins could interfere with the detection of metabolites in cultured astrocytes and consequently be removed using the Deproteinizing Sample Preparation Kit (Biovision, USA). A glycogen assay kit (Biovision) was used to detect glycogen levels in cultured astrocytes. ROS levels were determined using the ROS Detection Assay Kit (Biovision). A glucose-6-phosphate colorimetric assay kit (Biovision) was used to detect the glucose-6-phosphate levels. NADPH levels were determined with the NADPH Quantitation Fluorometric Assay Kit (Biovision). Glutathione levels were analyzed with the Glutathione Fluorometric Assay Kit (Biovision).
To determine the glycogen levels in the brain, the mice were decapitated, and brain tissues in the penumbra were immersed in liquid nitrogen. Then, the brain tissues (10 mg) were homogenized with 200 μL 30% KOH on ice, and the homogenates were boiled for 10 min to inactivate enzymes. The boiled samples were centrifuged at 12,000 rpm at 4 °C for 10 min to remove insoluble sediments. Then, the supernatant was ready for the assay using a glycogen assay kit (Biovision), and the results were normalized to the protein levels in homologous samples.

The ATP content was measured using an ATP Colorimetric/Fluorometric Assay Kit (Biovision, Milpitas, CA, USA). A portion (150 mg) of the samples was homogenized in 100 μL of the ADP assay buffer and centrifuged at 16,000× g for 10 min at 4 °C. For sample deproteinization and neutralization, the supernatant was treated with a Deproteinizing Sample Preparation Kit (Biovision). Extracts (50 μL) were added to 50 μL of a reaction mixture containing the ADP assay buffer, ADP converter, ADP probe, and ADP developer. After mixing and incubation for 30 min at room temperature in the dark, the absorbance was measured at 570 nm.

To determine lactate secretion by infected PMNs into the culture medium, a total of 10 × 10⁶ PMNs in triplicate were infected with either wild type, ΔT4SS, ΔlamA, the complemented lamA/C, or formalin-killed wild-type bacteria at an MOI of 1 for 1 h. Following infection, the culture medium was retained and cooled on ice and subjected to deproteinization using a deproteinizing sample preparation kit (BioVision) following the manufacturer’s instructions (104 (link), 105 (link)). The samples were analyzed for lactate using a lactate assay kit (Sigma) according to the manufacturer’s instructions with a Synergy H1 microplate reader (Biotek).