Cells were seeded and treated with or without myriocin in 6-well plates before being washed in cold PBS and collected by centrifugation. The levels of glucose, pyruvate, lactate, and citrate were detected by colorimetry methods using respective assay kits (Solarbio, BC2500, BC2200, BC2230 and BC2150) according to manufacturer’s instructions.
Bc2150
Manufactured by Solarbio
Sourced in China
The BC2150 is a laboratory centrifuge capable of achieving a maximum speed of 15,000 rpm and a maximum RCF of 21,000 x g. It is designed for a wide range of general-purpose applications in research and clinical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Bc2200, by Solarbio (3 mentions)
Bc2500, by Solarbio (1 mentions)
Bc2230, by Solarbio (1 mentions)
Ab204718, by Abcam (1 mentions)
Ab83431, by Abcam (1 mentions)
Bc2505, by Solarbio (1 mentions)
Bc1580, by Solarbio (1 mentions)
Uv 2600 spectrophotometer, by Shimadzu (1 mentions)
S0026, by Beyotime (1 mentions)
Bc0980, by Solarbio (1 mentions)
Bc2205, by Solarbio (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
5 protocols using bc2150
1
Metabolite Measurement in Cells
2
ACL Activity Assay with NADH Monitoring
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The ACL activity was determined as previously described with minor modifications [36 (link)]. In brief, the assay detects the ACL-catalyzed generation of oxaloacetate by coupling the oxidation of NADH catalyzed by malate dehydrogenase. The oxidation of NADH was monitored by the change in 340 nm light absorption, and ACL activity was calculated using the molar extinction coefficient of NADH (6.22 mM− 1 cm− 1). The ACL assay was conducted in total volume of 1 mL, containing 200 μL of extract, 20 mm MgCl2, 200 mM Tris-HCl (pH 8.4), 10 mM ATP, 1 mM DTT, 10 mM citrate, 0.2 mM CoA, 0.1 mM NADH and 6 units of malate dehydrogenase. Citrate were detected using citric acid (CA) content assay kit (Solarbio® BC2150).
3
Metabolic Profiling Assay Protocol
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4
Quantifying Pyruvic and Citric Acid
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The contents of PA and CA were measured at different time points (3, 5, 7, 9, 11 DAT) using pyruvic acid and citric acid assay kits (BC2200 and BC2150, respectively; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer’s instructions. Three independent biological replicates were assessed. All germinated seeds were rapidly frozen in liquid nitrogen and stored at −80 °C until analysis. The reaction mixtures were measured at 520 nm for PA and 545 nm for CA with a UV2600 spectrophotometer (Shimadzu). The PA content was calculated using a standard curve and expressed in µg g−1 (FW), while the CA content was calculated and expressed in µmol g−1 (FW).
5
Metabolic Analysis of Fungal Response to SDHIs
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The conidial suspensions were added to YEPD (1 × 105 conidia per 100mL YEPD) and incubated for 24 h, then the concentrations of five SDHIs were added to the cultures as described above. After incubation with an additive for 2 days, the mycelia were collected and used for the determination of pyruvic acid, acetyl-CoA, ATP, and citric acid. Pyruvic acid and acetyl-CoA were assayed using a pyruvic acid content test kit (Solarbio, BC2205, Beijing, China) and an acetyl-CoA content test kit (Solarbio, BC0980, Beijing, China), respectively. ATP and citric acid production were assayed using an ATP assay kit (Beyotime, S0026, Nanjing, China) and a citric acid content test kit (Solarbio, BC2150, Beijing, China), respectively. In short, 0.05g of mycelia were added to the corresponding lysis buffer of different detection kits. After the lysis of mycelia, pyruvic acid, acetyl-CoA, ATP, or citric acid production were determined according to the manufacturer’s instructions. The experiments were performed three times independently.
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