Calcein am

At 24 hpt, HUVECs were harvested and counted. Cells (5x10⁴ cells/well) were seeded in 1 ml of medium on a poly hydroxyethyl methacrylic acid (polyHEMA) coated 24-well plate (final density ≈1.3 mg of polyHEMA per cm²) and in a 24-well plate control. After 24 and 48 hours (48 and 72 hpt), cells were harvested and concentrated by centrifugation. Cell pellets were re-suspended in 100 μl of fresh medium and stained with calcein AM (Invitrogen) or WST-1 (Roche), according to the manufacturer’s instructions. After an hour, relative fluorescence units (RFU) for calcein AM (excitation/emission: 495/515 nm) or absorbance (A450-A650 nm) for WST-1 were read with a Modulus microplate reader (Promega). Results were presented as RFU or absorbance measurements in the polyHEMA-coated wells normalized to the control well, relative to mock-infected or negative-control mi/siRNA-transfected well.

Cytotoxicity of NK cells was assessed by lysis of K562 target cells, loaded with the
fluorescent dye calcein-AM (Life Technologies, Paisley, United Kingdom). K562 cells
were incubated and loaded with 10 µM calcein-AM for 30 min at
37°C, before washing in K562 maintenance media and serum-free media for 15
min. K562 cells and freshly isolated dNK cells or PB NK cells were cocultured in
serum-free dNK culture media in V-bottom, 96-well plates (Corning Life Sciences, The
Netherlands) for 4 h at ratios of 1:1–20:1 E:T. calcein-AM released into the
supernatant was assessed by use of a GloMax-Multi+ microplate spectrofluorimeter
(Promega, Southampton, United Kingdom) with excitation filter 485 and emission filter
530. Data are expressed as fold lysis over control, containing no NK cells but
matched numbers of target cells.

To evaluate the follicle density (FD) and to reduce heterogeneity, two 3mm biopsies (January 2018 to August 2018) or four 2mm biopsies (September 2018 to April 2020) from different cortex areas were collected for each patient [10 (link)]. Cortical biopsies were then incubated in a 5 % CO2 incubator at 37℃ for 2 hours in 0.8 mg/ml collagenase Type 1A (Sigma-Aldrich Chemie GmbH, Munich, Germany) supplemented with 0.2 % calcein-AM (Promega GmbH, Mannheim, Germany) and DPBS (Gibco by Life Technologies, Paisley, UK) for digestion and staining. After pipetting and placing at room temperature for 30 minutes, follicles descended to the bottom of the well so that FD could be measured by fluorescence and light microscopy. Primordial or primary follicles were identified by a single layer of flattened or cuboidal granulosa cells (GCs) [19 (link)]. Secondary preantral follicles were identified by two or more layers of GCs, as well as the absence of antrum [20 (link)]. The meander-shaped counting method was used to determine the number of follicles in each well [10 (link)]. To reduce the bias, follicles were blindly counted by two independent observers. Then we calculated the FD for every patient to make values comparable, converting the follicle numbers in 2 × 3mm or 4 × 2mm biopsies to the volume of one 3mm biopsy.