Multiplex electro chemiluminescence

Culture supernatants from the EpiVaginal tissues (apical), PBMCs (basal) and Vk2/E6E7 (with or without bacterial co-culture) were collected separately from various experiments. A custom designed 3-plex assay for GRO-α (CXCL1), MIP-3α (CCL20), and RANTES (CCL5) was used via Multiplex Electro-chemiluminescence (Meso Scale Discovery, USA) as per manufacturer's instructions.

Human MAPCs (n = 2) used in this study were isolated by ReGenesys BVBA (Athersys affiliate; Heverlee, Belgium) from the bone marrow of a 30-year-old female and a 45-year-old male volunteer, with informed consent and ethical approval. Isolation and culture of the cells was carried out as outlined in electronic supplementary material (ESM) Methods [22 (link)].
Phenotypic analysis of the human MAPCs was performed using fluorochrome-conjugated antibodies recognising cluster of differentiation (CD) 3, CD31, CD34, CD40, CD44, CD86, CD105, fetal liver kinase 1 (Flk1), HLA-ABC and HLA-DR (ebioscience, San Diego, CA, USA). Acquisition was done by using a Gallios multicolour flow cytometer (Beckman Coulter, Suarlée, Belgium). For analysis of the samples, FlowJo version 10.1 (Tree Star, Ashland, OR, USA) software was used.
Cell-free supernatant fractions were assayed for a pro-inflammatory, cytokine, chemokine, angiogenesis and vascular inflammation panel (see ESM Methods) by multiplex electrochemiluminescence (Meso Scale Discovery, Rockville, MD, USA) as per manufacturer’s protocol.
The angiogenic potential of human MAPCs was examined in the chick chorioallantoic membrane (CAM) as described [23 (link)].