R 1 hr at room temperature. After washing 3 times in PBS, slides

R 1 hr at room temperature. After washing 3 times in PBS, slides were mounted in medium containing DAPI (VectashieldH; Vector Laboratories; Burlingame, CA). Fluorescence was visualized on a Nikon Eclipse E-800 fluorescence microscope equipped with a spot digital camera.were calibrated against pH by the high-K+-nigericin technique [51,52].StatisticsStatistical analysis was performed using the Student’s unpaired t-test with the help 18325633 of a free online calculator (StudentsTTest.com). P,0.05 was considered significant.Supporting InformationFigure S1 Existence of distinct subpopulations of cells in primary cultures from older donors. (A,B) Phase contrast micrographs of primary cells from 56-year-old donor at passage 3. (C,D) Phase contrast micrographs of primary cells from 70-yearold donor at passage 1. Note that uniform subpopulations with polygonal cells growing in monolayers (B,D) were detected among non-dividing cells displaying signs of senescence (A,C) in primary cultures of both older donors. 200x. (TIF) Figure S2 Morphology of subconfluent HCEnC-21 and HCEnC-21T cells. Phase-contrast micrographs of HCEnC-21 (A) and telomerase-transduced HCEnC-21 (HCEnC-21T; (B) cells in the subconfluent state. (TIF) Figure S3 Immunofluorescence detection of ZO-1 in later passages of HCEnC-21 and HCEnC-21T. (A) Confluent monolayers of passage-32 HCEnC-21 and (B) passage-46 HCEnC-21T cells were fixed and labeled for ZO-1 (green) and nuclei (blue). (C) Representative example of the secondary antibody only controls showing no antibody binding. Note that ZO-1 in later passage cells localized to the cell-cell contacts as seen in earlier passage cells. 400x. (TIF)Transendothelial Resistance (TER)HCEnC-21 and HCEnC-21T cells were plated in FNC-coated, 12-well transwell inserts (1.12 cm2 growth area, 0.4 mm pore size) at a density of 100,000 cells per transwell. Medium was changed every other day. TER was measured every 2? days using the EVOM2 Epithelial VoltOhmMeter (WPI; Sarasota, FL) over the course of 4.5 wk. Immortalized human corneal epithelial (HCLE) cells were used as a positive control, whereas an empty FNCcoated (AthenaES) transwell served as a background control. Two to three different passages per group per cell type were analyzed. For data analysis, TER values for every time point were averaged and the SEM was calculated.Measurement of Proton-coupled Lactate UptakeLactate-dependent pH-tracing measurements were conducted as previously published [29], with minor modifications. In brief, HCEnC-21 and HCEnC-21T cells were cultured on a 0.2 mm pore size, rigid Anodisc filter (KDM5A-IN-1 site Whatman; Kent, UK) and incubated in bicarbonate-free Ringer solution containing 10 mM of the pH-sensitive fluorescent dye 2979-bis(2-carboxyethel)-5(6)carboxyfluorescein acetoxymethyl ester (BCECF-AM) at room temperature for 30 min. Anodisc filters were placed in a doublesided perfusion chamber [51] and lactate (20 mM) was applied for 20?5 sec to the apical and basolateral compartments by gravity flow (approximately 0.5 ml per min). The temperature was constant at 37uC throughout the measurement. Fluorescence was excited at both 495610 and 440610 nm, and emission was detected at 520?50 nm. Emission ratios were calculated for excitation at 495 and 440 nm (495/440). Ratios obtained at 1 HzAcknowledgmentsWe would like to thank Dr. David A. Sullivan for the generous gift of the 293GPG cells and the pBABE-puro-hTERT plasmid.Author ContributionsConceived and designed the experiments: TS.R 1 hr at room temperature. After washing 3 times in PBS, slides were mounted in medium containing DAPI (VectashieldH; Vector Laboratories; Burlingame, CA). Fluorescence was visualized on a Nikon Eclipse E-800 fluorescence microscope equipped with a spot digital camera.were calibrated against pH by the high-K+-nigericin technique [51,52].StatisticsStatistical analysis was performed using the Student’s unpaired t-test with the help 18325633 of a free online calculator (StudentsTTest.com). P,0.05 was considered significant.Supporting InformationFigure S1 Existence of distinct subpopulations of cells in primary cultures from older donors. (A,B) Phase contrast micrographs of primary cells from 56-year-old donor at passage 3. (C,D) Phase contrast micrographs of primary cells from 70-yearold donor at passage 1. Note that uniform subpopulations with polygonal cells growing in monolayers (B,D) were detected among non-dividing cells displaying signs of senescence (A,C) in primary cultures of both older donors. 200x. (TIF) Figure S2 Morphology of subconfluent HCEnC-21 and HCEnC-21T cells. Phase-contrast micrographs of HCEnC-21 (A) and telomerase-transduced HCEnC-21 (HCEnC-21T; (B) cells in the subconfluent state. (TIF) Figure S3 Immunofluorescence detection of ZO-1 in later passages of HCEnC-21 and HCEnC-21T. (A) Confluent monolayers of passage-32 HCEnC-21 and (B) passage-46 HCEnC-21T cells were fixed and labeled for ZO-1 (green) and nuclei (blue). (C) Representative example of the secondary antibody only controls showing no antibody binding. Note that ZO-1 in later passage cells localized to the cell-cell contacts as seen in earlier passage cells. 400x. (TIF)Transendothelial Resistance (TER)HCEnC-21 and HCEnC-21T cells were plated in FNC-coated, 12-well transwell inserts (1.12 cm2 growth area, 0.4 mm pore size) at a density of 100,000 cells per transwell. Medium was changed every other day. TER was measured every 2? days using the EVOM2 Epithelial VoltOhmMeter (WPI; Sarasota, FL) over the course of 4.5 wk. Immortalized human corneal epithelial (HCLE) cells were used as a positive control, whereas an empty FNCcoated (AthenaES) transwell served as a background control. Two to three different passages per group per cell type were analyzed. For data analysis, TER values for every time point were averaged and the SEM was calculated.Measurement of Proton-coupled Lactate UptakeLactate-dependent pH-tracing measurements were conducted as previously published [29], with minor modifications. In brief, HCEnC-21 and HCEnC-21T cells were cultured on a 0.2 mm pore size, rigid Anodisc filter (Whatman; Kent, UK) and incubated in bicarbonate-free Ringer solution containing 10 mM of the pH-sensitive fluorescent dye 2979-bis(2-carboxyethel)-5(6)carboxyfluorescein acetoxymethyl ester (BCECF-AM) at room temperature for 30 min. Anodisc filters were placed in a doublesided perfusion chamber [51] and lactate (20 mM) was applied for 20?5 sec to the apical and basolateral compartments by gravity flow (approximately 0.5 ml per min). The temperature was constant at 37uC throughout the measurement. Fluorescence was excited at both 495610 and 440610 nm, and emission was detected at 520?50 nm. Emission ratios were calculated for excitation at 495 and 440 nm (495/440). Ratios obtained at 1 HzAcknowledgmentsWe would like to thank Dr. David A. Sullivan for the generous gift of the 293GPG cells and the pBABE-puro-hTERT plasmid.Author ContributionsConceived and designed the experiments: TS.