Deproteinizing sample preparation kit tca

According to the instructions of the ATP Colorimetric Assay Kit (Abcam), 1 × 10⁶ s-generation BMSCs treated with different oxygen concentrations were dissolved in 100 μL of ATP Assay Buffer and centrifuged at 4 °C and 15,000 g for 8 min. The collected supernatant was deproteinized with the Deproteinizing Sample Preparation Kit—TCA (Abcam) to remove interfering enzymes. Next, 5 μL of enzyme-depleted supernatant was added to a 96-well transparent plate and made up to 25 μL/well with the ATP Assay Buffer. The standard well included 25 μL/well of standard dilution. Next, 22 μL of the ATP Assay Buffer was mixed with 1 μL of ATP Probe, 1 μL of ATP Converter, and 1 μL of Developer Mix into a 25-μL Reaction Mix and added to each well. The samples were mixed well and incubated at room temperature for 0.5 h protected from light before measuring the absorbance at an optical density (OD) of 570 nm.

Fifteen mg of tissue were washed in cold PBS twice. Tissues were homogenized in 100 μL of ATP Assay Buffer with a sonicator. Resulting suspension then was centrifuged for 4 minutes at 4°C at 13,000 x g and supernatant was transferred to a new tube. Samples were deproteinized and neutralized using the Deproteinizing Sample Preparation Kit–TCA (Abcam #ab204708). ATP concentration was measured using ATP assay kit (Abcam #ab 83355). Briefly, the ATP Reaction Mix was prepared and added to all standards and samples in a 96 well plate. Plates were incubated in a darkened container for 30 minutes at room temperature. Fluorescence was read using a microplate reader (SpectraMax Gemini XPS, Molecular Devices) with the excitation/emission setting at 485/590nm. The ATP concentration in the samples was calculated relative to the standard curve.

L-lactate was measured in cells and lung tissues using the L-lactate Assay kit (ab65331, Abcam) according to the manufacturer’s instructions. All samples were deproteinized with the Deproteinizing Sample Preparation Kit-TCA (ab204708, Abcam) before analysis. Protein concentration in samples before deproteinization was measured for normalization.

SH-SY5Y cells were pretreated with or without 0.5 mg/ml KSM-66 for 24 h and then incubated with 100 μM 6-OHDA for 2 h. For post-treatment, SH-SY5Y cells were incubated with 100 μM 6-OHDA for 2 h then incubated with 0.5 mg/ml KSM-66 for 24 h. Cells were collected and washed with PBS. ATP content was measured by a commercial ATP Assay Kit (Colorimetric/Fluorometric) (Abcam, UK) according to the manufacturer's instruction. Briefly, cells (1 × 10⁶) were lysed in 100 μl of ATP assay buffer, homogenized, and centrifuged (13,000 ×g, 5 min, 4 °C) to pellet insoluble materials. The supernatants were collected, transferred to new tubes and deproteinized using Deproteinizing Sample Preparation Kit – TCA (Abcam, UK). Briefly, 10 μl of cold TCA was added into 100 μl sample. The sample tube was placed on ice for 15 min and centrifuged at 12,000 x g for 5 min. The supernatant was transferred into another tube. Samples were diluted at 1:10 in ATP assay buffer. To obtain fluorescence measurements with a standard ATP, 50 μl of supernatant was mixed with 50 μl of ATP reaction mix solution. The standard curve of ATP was obtained by serial dilution of 0.1 mM ATP solution. The plates were incubated at room temperature for 30 min, while being protected from light and fluorescence in the wells was measured at Ex/Em = 535/587 nm using a multimode reader, Clariostar, BMG LABTECH.

Cardiac tissues were deproteinized using Deproteinizing Sample Preparation Kit–TCA (ab204708; Abcam), and the total nitrate/nitrite content was measured with Colorimetric Nitric Oxide Kit (ab65328; Abcam). Samples were run in duplicates following the manufacturer's instruction. The method was based on a two‐step process. The first step converts nitrate to nitrite using nitrate reductase. The second step converts nitrite to a deep purple azo compound using Griess reagents. A colorimetric microplate reader at 540 nm was used to measure the level of azo compounds reflecting the NO amount in the sample.

Naïve T cells were cultured under iTreg polarizing conditions for 24–48 hours before measuring lactate levels in the culture medium using a Lactate Assay Kit (Sigma-Aldrich, Cat # MAK064) and a ClarioStar plate reader. The samples were deproteinized to remove LDH and other proteins using a Deproteinizing Sample Preparation Kit-TCA (Abcam, Cat # ab204708) prior to lactate measurement. To observe absorption of lactate by fully differentiated Treg cells, Treg cells were differentiated under the stimulation of 1.25 μg/mL plate bound anti-CD3 antibody, 1 μg/mL soluble anti-CD28 antibody, 5 ng/mL active TGF-β, and 5 ng/mL IL-2 for 3 days. Differentiated Treg cells were cultured in 3 or 15 mM lactate for 24 hours, and the remaining lactate in the culture medium was measured by lactate assay.