Ham s f12 medium

We brought hEECs from the Procell Life Science & Technology Co., Ltd). The cells were cultured in Ham's F‐12 medium (Biological Industries, Kibbutz Beit Haemek) contained with 1% penicillin and streptomycin (Beyotime) and 12% fetal bovine serum (Gibco) in an atmosphere of 5% CO₂, at 37°C.
The hEECs were treated with 1 μg/mL LPS (L5293, Merck) for 3 h to establish an endometritis model in vitro.¹⁹

‘Plating medium’ was composed as follows: 10% newborn calf serum, 2 mM L-glutamine, penicillin/streptomycin (100 units/mL and 0.1 mg/mL respectively), 0.1 mg/mL gentamycin (Biological industries, Kibbutz Beit-Haemek, Israel) in Earl’s Medium 199 (Sigma). ‘Assay medium’ contained: 2 mM L-glutamine, penicillin/streptomycin (100 units/mL and 0.1 mg/mL respectively), 0.1 mg/mL gentamycin, 550 nM hydrocortisone (Sigma) in Dulbecco-modified Earl’s medium (DMEM) diluted 1:1 in Ham’s F12 medium (Biological industries, Israel). For growing hCMEC/D3 (brain endothelial cell line) EGM-1 medium was used: EGM-2 medium (Lonza) supplemented with FBS, bFGF, gentamycin, ascorbic acid and hydrocortisone according to the manufacture instructions (EGM-2 SingleQuots, LONZA).

The Chinese hamster ovary CHO-K1 cell line with a stable overexpression of the human NK1 receptor (hNK1R-CHO cells) were obtained as a gift from Dr. Attila Keresztes and Dr. John M. Streicher [24 (link)]. Cells were grown in Ham’s F12 medium (Biological Industries, Beit HaEmek, Israel) supplemented with 10% Foetal Bovine Serum (Biological Industries, Beit HaEmek, Israel) and 400 μg/mL G418 (Capricorn Scientific, Ebsdorfergrund, Germany), incubated in a humidified atmosphere with 5% CO₂ at 37 °C.
After reaching almost total confluence, the cells were washed with Dulbecco’s PBS (Biological Industries, Beit HaEmek, Israel), detached from the flasks using 0.05% trypsin-EDTA (Biological Industries, Beit HaEmek, Israel) at 37 °C, then diluted with medium and centrifuged for 10 min at 200× g. Obtained pellets were resuspended in the medium for the manual calculation of the cell quantity using Trypan Blue Solution (Biological Industries, Beit HaEmek, Israel) contrast.