Coulter flow cytometer

According to the manufacturer’s instructions, a cell cycle kit (Beckman Coulter) was used to assess the cell cycle in cultured cells using flow cytometry. While employing the recommended procedures, flow cytometry analysis (Beckman Coulter, Inc., Brea, CA, USA) was utilized to identify cell apoptosis in cultured cells using the Annexin V-FITC kit (BioVision, Milpitas, CA, USA). In brief, cells for all groups were cultured at 5 × 10⁵ cells/T75 flask and incubated overnight. After treatment with taxol (13.0 µg/mL) and CMC-AgNPs (7.9 µg/mL) or medium for 48 h, cells were allowed to grow in a 25 cm³ flask until they achieved 70–80% confluence. The cells were then rinsed in PBS and suspended at 5 × 10³–5 × 10⁶ cells/mL in 1 × binding buffer. Then, we added 100 μL of cell suspensions, 5 μL of dissolved PI, and 5 μL of annexin V-FITC solution, and incubated for 15 min in the dark. Following that, we added 400 μL of ice-cold 1 binding buffer and carefully mixed it. Flow cytometric analysis on a COULTER Flow Cytometer (Beckman Coulter) was used to identify apoptotic cells [54 (link),55 (link)].

The intracellular ROS levels were detected by ROS detection CM-H2DCFDA. After 8h treatment with Ad-mock, Ad-vp3, and Ad-vt, the cells were washed with PBS three times and stained with CM-H2DCFDA (10µM) for 30 min at 37 °C with shaking. The level of ROS was measured by a BECKMAN COULTER flow cytometer.

Blood was collected from patients in maintenance therapy group. Peripheral blood mononuclear cells (PBMCs) and immune cells were analyzed before to and following each cycle of the maintenance therapy, and then the difference in white blood cells (WBCs), lymphocytes, monocytes, B cells, T cells, and NK cells were compared. Human peripheral blood immune cells were analyzed by Coulter Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA). CD45⁺CD3⁺ is the T‐cell population, CD45⁺CD3⁻CD19⁺ is the B‐cell population and the cell population of CD3⁻CD56⁺/16⁺ is NK cells. The detailed protocol²⁷ and materials for flow cytometry are shown in Supplemental materials.

Flow cytometric analysis of stained cells was performed on a Beckman Coulter flow cytometer (Beckman Coulter, Fullerton, CA, USA). Dual staining of cells with Annexin V and PI was used to assess the percentages of apoptotic cells. Cells (1 × 10⁶) were washed in cold PBS and resuspended in 200 μl staining solution [10 mM 4-(2-hydroxyethyl)- 1-piperazineethane- sulfonic acid (HEPES) pH 7.4, 140 mM NaOH, and 2.5 mM CaCl₂] containing 5 μl of Annexin V-FITC and 10 μ of 20 mg/ml PI (BD Pharmingen, Franklin Lakes, NJ, USA) for 15 min at room temperature in the dark. Percentages of viable cells (double negative for Annexin V and PI), early apoptotic cells (Annexin V-positive and PI-negative cells), and late apoptotic cells (double positive for Annexin V and PI) were calculated. For cell cycle analysis, cells (5 × 10⁵) were washed in PBS, fixed with cold 70% ethanol, and incubated with 25 μg RNAse A (Sigma-Aldrich) for 1 h at 37 °C. Prior to analysis, 25 μl (5 mg/ml) of PI (Sigma-Aldrich) was added and the percentages of the cell population in sub-G1, G1, S, or G2/M phases were calculated from histograms.

For pre-G1 analysis, log-phase cells were treated for 48 h (A-1210477 or Vincristine), washed twice with PBS, permeabilized with 0.1% Triton X-100 and stained with Propidium Iodide. The apoptotic cells were evaluated as the pre-G1 proportions in the cell cycle. After 20,000 events were collected on a Beckman coulter flow cytometer, data were analyzed using CytExpert1.2 software.
Alternatively, for Annxin V and PI double staining assay, the cells were washed twice with PBS after harvest, and stained with Annxin V and PI in the buffer containing 10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4.