” [40]. For membrane integrity, two fluorescent stains were used, and for mitochondrial polarization a single two-color stain was used, and thus, two-color compensation of spectral overlap could be achieved simply by subtracting the signals on a linear scale for each detector. Compensation was first performed with manual

adjustments on the instrument itself and then double checked using the software “Kaluza v1.2” from the INCB024360 order manufacturer Beckman-Coulter. Fluorescence compensation was conducted individually for each type of experiment, once for membrane integrity (using Syto13 and ethidium bromide) and again for mitochondrial polarization (JC-1). However, the settings of fluorescence compensation were kept the same for each run throughout each experiment. It should be noted that the compensation conditions stated in the manuscript are specific Metformin in vitro to the fluorescent stains and instrumentation used in the investigation. The flow cytometer used in the investigation was subject to routine quality control runs in order to ensure accuracy of results. The instrument underwent routine monthly checks carried out using fluorescent beads purchased from the manufacturer. This research article meets the minimum information standard for flow cytometry experiments

(MIFlowCyt). The raw flow cytometry data is available in the Flow Repository (www.flowrepository.org – ID: FR-FCM-ZZ6W). Fig. 1 shows typical light scatterplots of the forward scatter (y-axis) and side scatter (x-axis) of HUVEC in suspension, either untreated HUVEC control, or after cells have been plunged into liquid nitrogen. Each dot on these plots represent a single event through the flow cytometer. Fig. 1A shows the raw unprocessed data of all events in room temperature controls, and depicts three populations, grouped into regions: R1 with high forward and high side scatter events (26%), R2 with low forward and however high side scatter events (6%), and R3 with low forward and low side scatter events (68%). Commonly, a threshold is established on the forward scatter channel under the assumption that this

threshold allows for the discrimination of cells from debris, where only events greater than the value of this threshold will be registered by the flow cytometer. Fig. 1B shows the same data as Fig. 1A, after application of a threshold on the forward scatter intensity, where events with forward scatter intensity below the threshold have been removed. Though debris makes up the majority of events in R3, this is not necessarily true for the events in R2. Fig. 1C shows a plot of forward versus side scatter for HUVEC without cryoprotectant directly plunged into liquid nitrogen to induce cryoinjury including the raw unprocessed data of all events. In Fig. 1C two populations R2 (32%) and R3 (68%) consist of the majority of events, with few events present in R1 (<1%). Fig.