2 7 dichlorofluorescein diacetate dcfda

Gold (III) chloride trihydrate was purchased from Sigma Aldrich (St. Louis, MO, USA). Sodium hyaluronate (50 kDa) was acquired from Lifecore Biomedical Inc. (Chaska, MN, USA). Potassium bromide (KBr), sodium hydroxide (NaOH), and paraformaldehyde were procured from Sigma Aldrich (St. Louis, MO, USA). Sodium chloride and potassium chloride were obtained from Sigma Aldrich (Bangalore, India). Disodium hydrogen phosphate was procured from Thermo Fischer Scientific (Mumbai, India). Potassium dihydrogen phosphate was acquired from Merck (Mumbai, India). Leibovitz (L-15) medium, Fetal bovine serum (FBS), trypsin, and penicillin-streptomycin were obtained from Gibco (Waltham, MA, USA). 2′,7′-Dichlorofluorescein diacetate (DCFDA) was purchased from Abcam (Cambridge, UK). A dead cell apoptosis kit with annexin V was procured from Invitrogen (Waltham, MA, USA). Propidium iodide was purchased from Sigma Aldrich (St. Louis, MO, USA). BODIPY C11 probe was purchased from Invitrogen (Waltham, MA, USA). Human recombinant CD44 protein was purchased from Invitrogen (Mumbai, India). Mouse monoclonal anti-CD44 antibody and goat anti-mouse IgG H&L (Alexa Flour 647) were purchased from Abcam (Cambridge, UK).

The intracellular ROS levels in M. aeruginosa were evaluated using an assay with the cell-permeant 2′7′-dichlorofluorescein diacetate (DCFDA; Abcam, Cambridge, UK), a fluorogenic probe which is deacetylated by cellular esterases to a non-fluorescent polar 2′7′-dichloro-dihydrofluorescein, which is readily oxidized by hydroxyl and peroxyl radicals and other ROS to a highly fluorescent dichlorofluorescein (DCF; Komosa et al., 2017 (link)). Microcystis cultures were collected by centrifugation and incubated with 25 μM DCFDA for 30 min in the dark, washed twice by centrifugation with fresh BG-11 medium to remove the excess of the probe, and then incubated with D. magna extracts as described in the “Experimental design” subsection. A 250 μl subset of each culture was transferred to a 96-well plate; the fluorescence of DCF was measured kinetically at 0, 5, 15, 30, 45, and 60 min following exposure to extracts using a Synergy HTX Multi-Mode Microplate Reader (BioTek, USA) at an excitation of 495 nm and emission of 528 nm. The results were normalized over the autofluorescence emission read at 680 nm (Thiel et al., 2017 (link)).