Chemotx 96 well plate

The ChemoTx^® 96-well plate (Neuroprobe, Gaithersburg, MD, USA) was used to assess motility of transfected OVCAR-3 and OV-90 cells. Addition of an even spread of dried 0.6 µL Geltrex (Gibco, Waltham, MA, USA) diluted 1:1 with media (RPMI1640 + 0.1% BSA) on the filter membrane was used to determine invasion. Briefly, cells were labelled with calcein AM (Life Technologies, Mulgrave, VIC, Australia) after 30 min of incubation in the dark on a nutator. Excess calcein AM were removed by washing the cells twice with media (RPMI1640 + 0.1% BSA). Portions of 4 × 10⁴ cells were then pipetted onto each pore of the filter above a microplate containing wells prefilled with chemoattractant (10% FCS) and media (RPMI1640 + 0.1% BSA). Reverse pipetting was employed at every step to prevent bubble entrapment. After a 6-hour, 37 °C incubation, cells that had migrated or invaded the filter were measured using the Triad series multimode detector (Dynex Technologies, Chantilly, VA, USA) at 485–520 nm. Assays were carried out in biological triplicate and had technical replicates to a total of n = 21–24 per cell line per construct transfected. Statistical analysis was carried out on GraphPad Prism 8 first by Shapiro−Wilk test of normality followed by either Mann−Whitney U test (non-normal distribution) or unpaired t-test (normal distribution).

De-immortalized DCs were seeded at 1*10⁵ cells/flask in 25 cm² cell culture flasks and allowed to settle overnight in a humidified atmosphere at 37 °C and 5% CO₂. Media were then removed and fresh media (5 ml) was given with either no additions (controls), NP conditions or classical activation scheme for 24 h. For migration, DCs (25*10³ in 25 µL volume were seeded on the membrane surface of 5 µm pore ChemoTx 96-well plate (NeuroProbe, Gaithersburg, MD) and incubated for 90 min at at 37 °C, with 10 ng/ml 6C-kine present in the bottom chamber, after which the number of migrated DCs in the bottom chambers were counted. To determine the IL-12p70-producing ability of migrated DCs, recombinant soluble CD40L was added directly to the bottom chambers, containing the migrated DCs, for 24 h, after which the IL12p70 was determined from the supernatant by ELISA (M1270, R&D Systems).