10 kd spin column

To determine flow-induced nitrate-nitrite, ATP or H₂O₂ release from the perfused mouse kidney, 500 µl of perfusate was collected. The perfusate was then centrifuged 10 min at 14 000 rpm and the supernatant added into a spin column with 10 kDa molecular weight cut-off filter for ultrafiltration (10KD Spin Column Abcam ab93349) and centrifuged at 10 000 rpm for 10 min. The centrifuged solutions was then used for nitrate-nitrite, ATP and H₂O₂ measurement.
Nitrate and nitrite levels in kidney perfusate were determined using a nitrate/nitrite fluorometric assay kit from Cayman Chemical (Nitric Oxide Metabolite Detection Kit Nb°780051) according to the manufacturer’s instructions.
Hydrogen peroxyde (H₂O₂) level was determined using the fluorimetric method of a hydrogen peroxide assay kit from Abcam (Hydrogen Peroxide Assay Kit Colorimetric/Fluorometric NbAb102500) according to the manufacturer’s instructions.
ATP level was measured using ATP determination kit from Invitrogen Molecular Probes (ATP Determination Kit Nb A22066) according to the manufacturer’s instructions.

Thorax muscle was freshly homogenized with 400 µl of NADH/NAD Extraction Buffer and centrifuged for 10 min at 4 °C at 10,000 × g. The supernatant was then added to the 10-kD spin column (Abcam, ab93349, USA) and centrifuged at 10,000 × g for 20 min at 4 °C. The filtrate was collected and used for NADH assay via the NAD/NADH Assay Kit (Abcam, ab65348, USA). The measured values were normalized to lysate protein levels.

NAD⁺ levels were measured using a NAD/NADH Assay kit (Cat# ab65348,Abcam, Cambridge, UK). All procedures were conducted strictly according to the manufacturers’ instructions. Approximately 20 mg of spinal cord tissue was digested in 400 μL NADH/NAD Extraction Buffer. After centrifuging in a 10 kD Spin Column (Cat# ab93349, Abcam) at 10,000× g for 10 min at 4 °C, half of the sample was transferred to a new tube and incubated at 60 °C for 30 min to decompose NAD⁺, while the remaining half was used as NADtotal (NADH plus NAD⁺). 20 μL of the NADtotal and 20 μL of the decomposed NAD⁺ sample were mixed with 30 μL Extraction Buffer and then incubated with 100 μL of Reaction Mix at RT for 5 min to convert NAD⁺ to NADH. After adding 10 μL of NADH Developer into each well, it was mixed. The reaction was allowed to cycle at room temperature for 20 min. The sample outputs were measured at OD 450 nm on a microplate reader in a kinetic mode.

4 μl serum was diluted in 96 μl ddH2O. The diluted sample was deproteinized using 10KD Spin column (Abcam, cat:93349). The solution was used to measure glucose level according to manufacturer’s instruction (Abcam, cat:65333).