Labeling autophagic vacuoles with monodansylcadaverine

Primary trophoblasts from NW and OB women were grown in DMEM-F12 (Sigma, D6429 and 51651C) with 10% FBS (Sigma, F2442) on sterile coverslips pretreated with 1% gelatin (Sigma, G1393) as an adhesion solution. At 66 h after plating, the ST were washed with phosphate-buffered saline (PBS; GIBCO, 70011–044) and incubated either in amino acid-free DMEM without FBS to trigger an autophagic response, or in full DMEM-F12 medium. After 6 h of incubation, syncytiotrophoblasts were washed twice with PBS and incubated with 50 μM monodansylcadaverine (Sigma, 30432) in PBS at 37°C for 1 h. The cells were then washed 3 times with PBS and analyzed immediately by fluorescence microscopy (excitation filter of 360 nm and an emission filter of 525 nm). The images were captured with a CCD camera, and MDC staining was quantified using ImageJ (National Institutes of Health). After visualization of MDC, cells were fixed, permeabilized, blocked by 1% BSA (Sigma, A2153) in PBS, and counter-stained with Alexa Fluor 568 Phalloidin (Thermo Fisher, A20184), a high-affinity filamentous actin probe.