Facscalibur cell sorting system

Lung cancer cells and BMCs were immobilized at 4°C for 10 h using 70% and absolute ethyl alcohol, respectively. Cells were washed with phosphate buffer solution. RNase A was added (final concentration: 50 μg/ml) to digest RNA at 37°C for 30 min. Propidium iodide solution was added (final concentration: 65 μg/ml) to stain cells for 30 min. Then, FACSCalibur™ Cell Sorting system (BD Biosciences; Franklin Lakes, NJ, USA) was utilized to perform flow cytometry to determine cell cycle. Based on cell cycle information, proliferation index was calculated using the following formula: $\text{Proliferation index}=\frac{S+G_2/M}{G_0/G_1+S+G_2/M}\times 100\%$
In this formula, G₀ /G₁, S and G₂ /M represent the relative numbers of cells in phase G₀ /G₁, S and G₂ /M, respectively.

Lung tissue samples were cut into pieces and placed in tubes with trypsin and collagenase buffer. The tissue samples were digested for 1 hour at 37°C, and the cells were then collected by centrifugation. Splenocytes were directly ground on 200-mesh steel filters. Red blood cells were then removed using alysis solution, and the cell concentration was adjusted to 1×10⁷/ml. A total of 100 μl of cells at a concentration of 1×10⁷/ml were recovered from BALF or tissue samples. Next, 10 μl of blocking buffer was added to the cells, which were then incubated for 15 min on ice. After being washed, the cells were incubated with 20 μl of FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 or control mouse IgG2b for 1h on ice in the dark. Then, the cells were washed with PBS and fixed in PBS containing 1% paraformaldehyde. The cells were either analyzed using a BD Biosciences FACS Calibur Cell Sorting System or were stored at 4°C in a refrigerator for subsequent testing. Flow cytometry analysis of various cell fractions was performed using FlowJo software.