Ab181421

The levels of alanine aminotransferase (ALT) (ab234579, Abcam Inc., Cambridge, MA, USA), aspartate aminotransferase (AST) (ab263883, Abcam), IL‐6 (ab178013, Abcam), TNF‐α (ab181421, Abcam), IL‐1β (ab217608, Abcam) and IL‐18 (ab215539, Abcam) was detected using ELISA kits. Briefly, the sample to be tested (containing antibody) was bound to antigen, and then, the labelled enzyme was bound to the complex to form antigen‐antibody‐labelled enzyme complex. The enzyme substrate was added to produce coloured product, and its optical density value was determined by spectrophotometer.

1 x 10⁴ cells were seeded in 6 well plate for all the groups (control, stress and A. indica Preconditioned). The cells were lysed using RIPA buffer containing Protease Inhibitor (Roche cat# 04693116001). The cell lysates were assessed for inflammatory cytokines and transcription factors using ELISA. The ELISA for IL-1 (Abcam cat # ab46052), TNF-α (Abcam cat # ab181421), NF-kappa (p65) (Abcam cat # ab133112) and IKKα and IKKβ (Cytoglow cat # CB5358) was performed using a commercially available kit, according to the manufacturer’s instructions.

From all individuals, five milliliters of intravenous blood were obtained after 8–12 h of fasting. Serum was obtained from whole blood by centrifugation at 2000× g for 10 min. Serum samples were then collected and stored at −80 °C until further analysis. The following methods measured serum inflammatory and antioxidant biomarkers by approved laboratories. Hs-CRP levels were measured by the hypersensitivity method using an ELISA kit (hs-CRP kit provided by DRG International Inc., Springfield, NJ, USA). The minimum detectable concentration of the abovementioned kit was about 0.1 mg/L. As recommended by the American Heart Association, the cut-off level for inflammation regarding hs-CRP was 3 mg/dL. According to the manufacturer’s instructions, serum levels of ILs and TNF-α were assessed using relevant commercial ELISA kits (ab181421, ABCAM; Trumpington, Cambridge, UK). MDA and TAC assay kits (Teb Pazhouhan Razi, Tehran, Iran) were used to measure serum antioxidant capacity. Rapid insulin ELISA kit (Monoband) and hemoglobin A1C (HbA1c) enzymatic kit (Pishtazteb, Iran) were used to measure biochemical markers of glucose metabolism.

RPMI 1640 medium (61870-036) and fetal bovine serum [FBS] (10099141) were purchased from Gibco (USA, NY). CORT and neferine (HY-N0441) were purchased from MedChem Express (NJ, USA). The GenElute Gel Extraction Kit (NA1111) was purchased from Sigma-Aldrich (Darmstadt, Germany). BamHI (R0136S) and XhoI (R0146S) were purchased from NEB (NY, USA). Lipofectamine 3000 Transfection Reagent (L3000015) was purchased from Invitrogen (CA, USA). G418 (G8161) was purchased from Solarbio (Beijing, China). RNApure Tissue and Cell Kit (CW0584), HiFiScript cDNA Synthesis Kit (CW2569), and Super TaqMan Mixture (CW2698) were obtained from Cwbiotech (Beijing, China). Antibodies against Bcl-2 (ab32124), Bax (ab32503), Bad (ab32445), p53 (ab26), Bak (ab32371), succinate-CoA ligase GDP-forming beta subunit [SUCLG2] (ab96172), aconitase 2 [ACO2] (ab110321), malate dehydrogenase 1 [MDH1] (ab180152), citrate synthase [CS] (ab96600), isocitrate dehydrogenase [IDH] (ab172964), NF-κB p65 (ab16502), and glyceraldehyde 3-phosphate dehydrogenase [GAPDH] (ab8245) were purchased from Abcam (Massachusetts, US). ELISA kits for IL-1β (ab100562), IL-2 (ab174444), IL-6 (ab46027), TNF-α (ab181421), interferon-γ [IFN-γ] (ab46025), and granulocyte colony-stimulating factor [G-CSF] (ab100524) were purchased from Abcam. Rabbit and mouse secondary antibodies were purchased from ECL.

ELISA was performed using the ELISA kits for TNF-α and IL-1β (ab181421 and ab214025; Abcam, USA) in order to detect the concentrations of TNF-α and IL-1β in culture supernatant of PBECs after centrifugation 12,000×g for 10 min at 4 °C. Absorbance at 450 nm was determined via a microplate reader according to manufacturer’s protocols.

TNF-α ELISA kits (ab181421, Abcam, UK), IL-8 ELISA kit (ab46032, Abcam, UK) and IL-1β ELISA kit (ab214025, Abcam, UK) were used to measure the cell TNF-α, IL-8 and IL-1βfollowing the instructions.