2.1. Blood draw and staining protocol
Fasting blood (50 ml) was drawn from healthy participants of an ongoing clinical study. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll–Hypaque (Sigma-Aldrich, St. Louis, MO) gradient centrifugation, washed in PBS (Dulbecco's Phosphate Buffered Saline, PBS without Mg++ and Ca++, Invitrogen-Biosource, Carlsbad, CA) and then incubated for 15 min at 4 °C with fluorescent-conjugated monoclonal antibodies: anti-CD3-PB, anti-CD4-FITC, anti-CD28-PE, anti-CD19 APC-Cy7 (all from BD biosciences, San Jose, CA) and anti-CD8-ECD (Beckman Coulter, Miami, FL). Stained cells were sorted on a FACS Vantage DiVa II (BD Biosciences, San Jose, CA) into the following fractions: CD4+T cells (CD3+CD4+), CD8+CD28+T cells (CD3+CD8+CD28+), CD8+CD28− T cells (CD3+CD8+ CD28−) and B cells (CD3−CD19+). The cell sorter was configured with 3 lasers, and argon-ion at 488 nm, a krypton at 647 nm, and a violet enhanced krypton at 407 nm; each laser outputting 200mW. An aerosol management system (Cytek Development, Inc., Fremont, CA) was used for operator protection. Cells were collected into AIM V serum-free media (Invitrogen, Carlsbad, CA). Sorted cells were pelleted by centrifugation at 7000 rpm at 4°C for 5min in an Eppendorf refrigerated microcentrifuge (Model 5417R, Eppendorf, Westbury, NY) and washed twice with cold PBS. Two aliquots of cell pellets, each containing approximately 0.5 million cells, were saved to prepare DNA for telomere length measurement and telomerase activity assay respectively. DNA was prepared using Gentra Puregene Cell kit (QIAGEN, Valencia, CA). Extracts for measuring telomerase activity were prepared from cell pellets according to the TRAPeze kit (Upstate/CHEMICON, Temecula, CA). Due to the low percentages of CD8+CD28− and B cells, not enough cells were obtained for telomere length measurement and/or telomerase activity assay in some participants, leading to various sample sizes.
2.2. Assay of telomere length
The telomere length measurement assay was adapted from the published original method (Cawthon, 2002
). The primers for the telomere PCR were tel1b
GTT-3′], used at a final concentration of 100 nM, and tel2b
CCT-3′], used at a final concentration of 900 nM. The primers for the single-copy gene (human beta-globin) PCR were hbg1
[5′ GCTTCTGACACAACTGTGTTCACTAGC-3′], used at a final concentration of 300 nM, and hbg2
[5′-CACCAACTTCATCCACGTTCACC-3′], used at a final concentration of 700 nM. The final reaction mix contained 20 mM Tris–HCl, pH 8.4; 50 mM KCl; 200 µM each dNTP; 1% DMSO; 0.4× Syber Green I (Invitrogen, Carlsbad, CA); 22 ng E. coli
DNA (MP Biomedicals, Solon, OH); 0.4 Units of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) and 0.5–10 ng of genomic DNA per 11 µl reaction. Tubes containing 26, 8.75, 2.9, 0.97, 0.324 and 0.108 ng of a reference DNA (from Hela cancer cells) were included in each PCR run so that the quantity of targeted templates in each research sample could be determined relative to the reference DNA sample by the standard curve method. The same reference DNA was used for all PCR runs.
All PCRs were carried out on a Roche Lightcycler 480 real-time PCR machine with 384-tube capacity (Roche Diagnostics Corporation, Indianapolis, IN). The telomere thermal cycling profile consisted of: cycling for T (telomeric) PCR: denature at 96 °C for 1 s, anneal/extend at 54 °C for 60 s, with fluorescence data collection, 30 cycles; cycling for S (single-copy gene) PCR: denature at 95 °C for 15 s, anneal at 58 °C for 1 s, extend at 72 °C for 20 s, 8 cycles; followed by denature at 96 °C for 1 s, anneal at 58 °C for 1 s, extend at 72 °C for 20 s, hold at 83 °C for 5 s with data collection, 35 cycles.
To control for inter-assay variability, 8 control DNA samples were included in each run. In each batch, the T/S ratio of each control DNA was divided by the average T/S for the same DNA from 10 runs to obtain a normalizing factor. This was done for all 8 control samples and the average normalizing factor for all 8 samples was used to correct the participant DNA samples to obtain the final T/S ratio. The T/S ratio for each sample was measured twice. If the duplicate T/S value and the initial value varied by more than 7%, the sample was run a third time and the average of the two closest values was reported. Typically, about 5% of samples needed to be assayed the third time. Using this method, the inter-assay coefficient of variation (CV) for telomere length measurement was 4%.
2.3. Gel-TRAP assay
Gel-TRAP assays were performed by the Telomerase Repeat Amplification Protocol (TRAP) using a commercial kit (TRAPeze Telomerase Detection Kit, Upstate/CHEMICON, Temecula, CA). Cells sorted by a FACS Vantage DiVa II were spun down, washed with DPBS and stained by Trypan blue, live cells were counted using a hemocytometer. 5 × 105 – 1 × 106 cells per sample were pelleted and lysed with 1 × CHAPS buffer as directed by the manual for the TRAPeze kit. An extract corresponding to 5000 cells/µl was made and between 2000 and 10,000 cells were used for TRAP reactions. The reaction was carried out according to the TRAPeze kit manual and run on a 10% polyacrylamide–8 M urea sequencing gel. The gel was exposed to a phosphorimager plate overnight and scanned on a STORM 860 (GE Healthcare, Piscataway, NJ). The 293T cell line was used as a positive telomerase activity control and standard. Telomerase activity is expressed as the equivalent number of 293T cells. Telomerase activity was quantified using the software ImageQuant 5.2 (GE Healthcare, Piscataway, NJ). Briefly, signals from the product ladders on the gels were added and normalized against the signal from internal control band for the same lane to get the product/internal control value. For each telomerase activity assay reaction, the product/internal value is divided by the product/internal control value from twenty 293T cells and then multiplied by 20 to obtain the final telomerase activity units, defined as 1 unit = the amount of product from one 293T cell/10,000 immune cells.
2.4. Participant description
The study participants were recruited from the San Francisco Bay Area, using flyers, advertisements, and notices in dementia clinics and senior daycare centers. The advertisement for volunteers requested healthy non-smoking women. Since data indicate that stress affects cell aging, we examined a sample with a range of stress levels. The sample included women who were caregiving for a family member, and control women, who were not caregiving. There were 34 dementia caregivers, 3 mothers of children with autism, and 29 control women, who were not caregiving. All participants filled the written consent to this study. They study was approved by UCSF Institutional Review Board for Human Research. Analyses by stress levels are beyond the scope of this paper, and will be reported elsewhere. The sample was examined as a whole, since the questions addressed here were independent of age and stress level. The sample was mostly white, and completed high school, with a mean age of 61, and mean BMI of 26.4 (See for demographics).
Demographic factors and body mass index a among 66 b study participants.
2.5. Statistical analysis
Means and standard deviations or frequencies for several demographic variables and BMI were computed across the sample (see ).
Because our data included multiple values per person for percent cell type, telomere length, and telomerase activity, mixed linear models (SAS PROC MIXED) with an unstructured covariance matrix were used to compute within-person correlations. We evaluated raw means and standard deviations for telomere length, telomerase activity, and percent cell types (SAS PROC MEANS) but used mixed models to compare within-person means for these variables, using F-tests of fixed effects to evaluate whether group values differed as a whole and contrast statements to test whether values for specific cell types differed from other groups (–). We also performed post-hoc pairwise tests of least-square means for the cell types, using the Tukey method to account for multiple comparisons. We used Spearman correlations to evaluate associations between age and cell variables.
Telomerase activity (per 10,000 cells) by cell type.
Percentage of each cell type.
Scatter plots of telomerase activity comparing EDTA and heparin collection tubes, and comparing morning and afternoon blood draws were created using Microsoft Excel. Box plots for telomerase and telomere length were generated using GraphPad Prism®.