Hipo mpp 96 microplate photometer

96-well Nunc Maxisorp™ flat-bottom plates (Invitrogen, Carlsbad, CA, USA) were coated overnight at 4 °C with either recombinant HER2 (aa23-652, GenBank ID: NP_004439) or VAR2CSA (ID1-ID2a, FCR3 strain, GenBank ID: GU249598). HER2 coat protein was produced like its Catcher-counterpart [45 (link)], while VAR2CSA is described elsewhere [49 (link)]. Plates were blocked for one hour in 0.5% skim milk powder in PBS at room temperature (RT). Sera were diluted 100× and added to the well in threefold dilutions in blocking buffer and incubated for 1 h at RT. Plates were washed with 1xPBS three times in between steps. Plates were incubated for 1 h at RT with HRP-conjugated secondary antibodies targeting either total mouse IgG, IgG1, IgG2a or IgG2b (Invitrogen, Carlsbad, CA, USA). Plates were developed with TMB X-tra substrate (Kem-En-Tec, Taastrup, Denmark). OD₄₅₀ was measured with a HiPo MPP-96 microplate photometer (Biosan, Riga, Latvia).

Parathyroid hormone (PTH) concentrations were measured using fetal plasma. Commercial kits from MyBioSource (San Diego, US), with washing performed on a Biosan plate washer (3D-IW8, Inteliwasher, Biosan, Riga, Latvia) were used as previously described (Usuda et al., 2017 (link)). Standards (calibration curve R² > 0.97) were assayed in triplicate (average coefficient of variation 6.1%) and samples were assayed in duplicate. The sensitivity in the assay was 1 pg/ml. Assays were performed in accordance with the manufacturer’s instructions, with absorbance at 450 nm read on a HiPo MPP-96 microplate photometer (Biosan, Riga, Latvia).

Sorted CD142^low and CD142^high fibroblasts (100,000 cells/well, 48-well plate) were cultured for 2 days, FBS was removed from the medium and cells were further cultured for 3 days. Cell debris was removed from the conditioned medium by centrifugation (300× g, 5 min), and HGF was detected with ELISA (Bio-Techne, Minneapolis, MN, USA, DHG00B) according to the manufacturer’s protocol. We measured the OD values of the plates on a HiPo MPP-96 Microplate Photometer (Biosan, Riga, Latvia).