Multiskan ascent elisa reader

The primary normal cell line (HDF-n) and three cancer cell lines (HT29, MDA-MB231, and SW780) were tested for viability after electroporation. These cell lines were chosen to compare the normal cell line with a few of the cancer cells lines that had shown a clear difference in membrane repair.
After harvesting, cells were washed in HEPES buffer (10 mM HEPES, 250 mM sucrose, and 1 mM MgCl₂ in sterile water) and diluted to 5.5 × 10⁶ cells/ml HEPES buffer. In 4 mm cuvettes with aluminum electrodes (Molecular BioProducts, Inc.) 300 µl cooled cells (8 °C) were electroporated by delivering 8 pulses of 100 µs, 1.2 kV/cm, and 1 Hz using a BTX T820 square wave electroporator. After 20 min incubation at 37 °C and 5 % CO₂ cells were diluted in culture medium to 3.1 × 10⁵ cells/ml and seeded in 96-well plates (100 µl/well). One day after treatment viability was measured by MTS assay (Malich et al. 1997 (link)) using Multiskan-Ascent ELISA reader (Thermo Labsystems).

Macrophage inflammatory protein- (MIP-) α (also chemokine (C-C motif) ligand 3 or CCL3), chemokine (C-C motif) ligand 5 (CCL5 or RANTES), and interleukin- (IL-) 6 concentrations were determined using ELISA kits purchased from Abcam Inc. (Cambridge, UK) with the appropriate matched antibodies according to manufacturer's instructions. Optical density at 450 nm was read on a Multiskan Ascent ELISA Reader (Thermo Labsystems, Helsinki, Finland).

Hemolymph phenoloxidase (PO) activity was determined using 2 μl of hemolymph obtained from each individual larva as previously described [34 (link)]. Hemolymph was collected, immediately diluted 1:10 in ice-cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH7.4) and centrifuged at 16,000 x g for 3 min at 4°C to obtain cell-free plasma. Samples were then snap frozen in liquid N₂ and conserved at -80°C until use. PO activity was assayed by adding 8 μl of cell-free plasma to 200 μL of 1 mM L-Dopa (Sigma-Aldrich) in PBS. Measurements were taken by an ELISA Multiskan Ascent reader (Thermolab Systems) at 490 nm every minute throughout 45 minutes. Since the observed absorbance curve was linear from 20–45 min after adding the substrate, the slope of the curve from 25–45 min of the reaction was used to calculate the absorbance increase per min. Measurements were repeated twice for each sample.

W. chondrophila antibodies in the serum samples were measured by ELISA, as described previously [10 (link),25 (link)]. The ELISA uses enriched outer membrane proteins isolated from purified elementary bodies of W. chondrophila as antigens. Optical densities (OD) were measured using the ELISA Multiskan ascent reader (Thermo scientific, Zurich, Switzerland) at 492 nm, against 650 nm as reference. All experiments were performed in duplicate. Control sera were included as reference to calculate ROC curves and cut-off levels for positivity, negativity, and grey zone. Details concerning the control sera and defining the cut-off levels can be found in the paper of Lienard et al., in which they describe the development of the W. chondrophila-specific ELISA [25 (link)]. Samples from the MUMC+ cohort were measured in three subsets, cut-off values for seropositivity were set at 0.395, 0.359 and 0.737. Seropositivity cut-off levels for the UMCG cohort were set at 0.164. The highest 10% of W. chondrophila-positive OD value ratios were selected as a high W. chondrophila. These ratios were determined by dividing the OD values by their respective cut-off value.