Sodium phosphate

All the reagents including Folin–Ciocalteu reagent, 1,1 diphenyl-2-picrylhydrazyl radical (DPPH), butylated hydroxyl toluene (BHT) and gallic acids were purchased from Sigma chemicals (St. Louis, MO, USA). Chemicals and solvents comprising sodium phosphate, anhydrous sodium carbonate, sodium hydroxide, ferric chloride, methanol, ethanol, ferrous chloride, potassium ferricyanide, sulfuric acid, sodium phosphate, sodium bicarbonate, sulfo-salicylic acid, trichloroacetic acid, orthophosphoric acid, ninhydrin, toluene, glacial acetic acid and boric acid were of analytical grade and procured from Merck (Darmstadt, Germany). Ultra-pure water (Victor diagnostic laboratories, Pakistan) was used as solvent during in vitro assays.

Streptavidin from Streptomyces avidinii, horseradish peroxidase (HRP), lysozyme, imidazole, Tris powder, 1-fluoro-2,4-dinitrobenzene (FDNB), phenylmethylsulfonyl fluoride (PMSF), sodium bicarbonate, sodium carbonate, sodium phosphate, sodium periodate, and sodium borohydride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ampicillin and isopropyl β-d-1-thiogalactopyranoside (IPTG) were purchased from LPS Solution (Daejeon, Republic of Korea). All enzymes for plasmid construction were purchased from Enzynomics (Daejeon, Republic of Korea). Pyridoxal phosphate and phosphate buffered saline (pH 7.4) were obtained from AMRESCO (Solon, OH, USA), and ethylene glycol was purchased from Junsei Chemical (Tokyo, Japan). Bovine serum albumin (BSA) was purchased from Roche (Basel, Switzerland). The bicinchoninic acid (BCA) protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was purchased from Kementec (Taastrup, Denmark). The single-stranded DNA aptamer that specifically binds to PfLDH called ‘2008s’ (5′-CTGGGCGGTAGAACCATAGTGACCCAGCCGTCTAC-3′) was synthesized by Integrated DNA Technologies (Singapore, Republic of Singapore), and biotin or HRP was conjugated at 3′-end of aptamer, respectively. Human LDH (hLDH) was purchased from Abcam (Cambridge, UK).

Cells were fixed for 10 min at room temperature in 1% formaldehyde, washed with PBS and incubated at 37°C for 16 h in the absence of CO₂ with freshly made staining solution: 0.3 mg/ml of 5-bromo-4-chloro-3-in dolyl-β-d-galactopyranoside (X-Gal; Promega Corporation; cat. no. V394A), 40 mM citric acid (Sigma-Aldrich; Merck KGaA), 40 mM sodium phosphate (Sigma-Aldrich; Merck KGaA) [stock solution (400 mM citric acid, 400 mM sodium phosphate) held at pH 6], 5 mM potassium hexacyanoferrate (Sigma-Aldrich; Merck KGaA), 5 mM potassium ferricyanide (Sigma-Aldrich; Merck KGaA), 150 mM NaCl (Sigma-Aldrich; Merck KGaA) and 150 mM MgCl₂ (Sigma-Aldrich; Merck KGaA). SA-β galactosidase staining was observed using a light microscope (Life Technologies; EVOS XL Core) and images were captured at ×40, ×100 and ×200 magnification in different areas in examples of each condition (31 (link)).