Fc500 flow cytometer

Telomere length of granulocytes and lymphocytes was analyzed in 105 samples from healthy donors as described before [17 (link)], 70 independent patients with AA, and 18 independent patients with DKC. In 5 AA patients and 1 DKC patient, telomere length measurement of the granulocytes was not possible due to insufficient cell number. Flow-FISH for telomere length was performed as described in detail before [12 (link), 59 (link)]. In brief, samples were mixed with a FITC-labeled or Alexa488-labled telomere-specific (CCCTAA)3-peptide nucleic acid FISH probe (Panagene, Daejeon, South Korea) for DNA hybridization followed by DNA counterstaining with LDS 751 (Sigma Aldrich, St. Louis, MO, USA). An FC 500 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) was used for data acquisition. Bovine thymocytes were used as internal controls to calculate telomere length in kilobases and samples were measured in triplicates. The cow thymocytes as well as granulocytes and lymphocytes from human samples were identified based on forward scatter and LDS 751 binding to double-stranded DNA. Telomere age was estimated by linear regression on the age-adjusted samples from healthy donors. Mean average error [MAE] was calculated as follows:
$$\mathrm{MAE}=\frac{1}{n}{\sum }_{i=1}^n\left|x_i\right|$$
with x = delta age. Mean delta age ( $\overline{x}$ ) was calculated as follows:
$$\overline{x}=\frac{1}{n}\sum _{i=1}^n{x}_i$$

The U87MG or A172 cells were seeded in 6-well plates at a density of 1× 10⁶ cells/well. The cells were treated for 24 h with 1 mM melatonin or 10 mM 3-MA. Afterwards, the cells were washed in PBS and stained with FITC-conjugated Annexin V and propidium iodide (BD Biosciences) in binding buffer. The cells were incubated at room temperature for 15 min in the dark and analyzed by a Beckman Coulter FC500 flow cytometer (Beckton Dickinson, Franklin Lakes, NJ, USA) using the WinMDI2.9 software program.

To confirm whether C. orbiculata induces apoptosis in HCT116, OE33, and KYSE70 cancer cells, the FITC Annexin V/Dead cell Apoptosis kit (Invitrogen, ThermoFisher Scientific) was used. Cells (15 × 10⁴ cells/mL) were seeded in 6 well plates and treated with varying concentrations of the crude extract of C. orbiculata for 48 h. Cells were harvested, washed two times with ice cold PBS and adjusted at a density of 1 × 10⁶ cells/sample (500 μL). The cells were stained with FITC Annexin-V/PI staining kit according to the manufacturer's instructions. The cells were analyzed using Becton Dickinson and Company (FC500) flow cytometer. Results were analyzed using Kaluza analysis 1.5 software.