Ph fix strips

Mice were vaginally infected as previously described (Enjalbert et al., 2009 (link); Pericolini et al., 2015 (link), 2017 (link)). Mice were maintained under pseudoestrus condition by subcutaneous injection of 0.2 mg of estradiol valerate in 100 μl of sesame oil (Sigma-Aldrich) 2 days prior to infection and 1 day after infection. Mice anesthetized with 2.5–3.5 (v/v) isoflurane gas were infected with 10 μl of 2 × 10⁹ cell/ml of CA-105 or CA-67 strains. Cell suspensions were administered with a mechanical pipette inserted into the vaginal lumen, close to the cervix. To favor vaginal contact and adsorption of fungal cells, mice were held head down for 1 min following inoculation. Mice were then allowed to recover for 24 h, during which Candida infection was established. The vaginal pH was measured with vaginal swab by pH-Fix strips (Macherey-Nagel GmbH & Co. KG, Germany) in each mouse before (day −2) and after (day 0) subcutaneous injection of estradiol valerate and at the end of experiments (day +3).

Three vaginal swabs were collected from each woman enrolled in this study. One swab was used for pH measurement using pH-Fix strips (Macherey-Nagel GmbH & Co. KG, Düren, Germany).
One swab was soaked in 1 ml of saline. A certain volume of this sample was cultured on BD Columbia CNA Agar containing 5% Sheep Blood (Becton Dickinson, New Jersey, USA). For G. vaginalis identification, mass-spectrometry (MALDI-TOF MS) analysis (Bruker Daltonik GmbH, Bremen, Germany) was performed. The remaining volume of the sample was used for Gram stain (Nugent’s criteria) for the diagnosis of BV. The last swab was soaked in 1 ml of saline and vortexed for at least one minute. Around 100 μL of the sample was examined under a light microscope (Olympus, Milan, Italy) to evaluate the presence of neutrophils (PMNs) and EC exfoliation. The numbers of PMNs and ECs were counted in four fields at ×400 magnification and expressed as the average number of PMNs or ECs/field, as previously described^{10 (link),19 (link),27 (link)}.
The remaining sample (900 μl) was centrifuged at 1,600 rpm for 10 min, and the cellular fraction was used for flow cytometric analysis or for assessment of EC damage or EC apoptosis.
Due to the limited amount of ECs in our samples, we were unable to perform all analyses for all samples. In each figure, we have reported the number of BV and healthy specimens used.