Five microlitres of buffy coat from all blood samples were smeared in 96-well plates. The cells were dried at room temperature for 20 min. Next, 100 µl of 3.7% formaldehyde in PBS was added to the sample for 20 min at room temperature to fix WBCs to assess morphology. The cells were washed 5 times with 100 µl of PBS. The cells were permeabilized with 2% Triton X solution for 10 min at room temperature. All antibodies, including 5 primary antibodies for WBCs (anti-CD45+ primary mouse antibody (ab8216) for leukocytes, anti-CD15+ primary mouse antibody (ab20137) for polymorphonuclear cells (neutrophils, eosinophils and basophils), anti-CD3+ primary mouse antibody (ab8671) for T lymphocytes, anti-CD19 + primary mouse antibody (ab31947) for B lymphocytes, and anti-CD14+ primary mouse antibody (ab182032) for monocytes), 5 primary antibodies for candidate proteins (anti-STAU2 primary rabbit antibody (ab184009), anti-AZI2 primary rabbit antibody (ab232654), anti-LMAN1 primary rabbit antibody (ab125006), anti-MMP9 primary rabbit antibody (ab38898) and anti-PLOD1 primary rabbit antibody (ab2647)) and 2 secondary antibodies (goat anti-mouse secondary antibody-Cy3 (ab97035) and goat anti-rabbit secondary antibody-FITC (ab6717)) (Abcam Co., Ltd., Cambridge, England) were prepared in 1:1000 dilution with binding buffer (2% FBS and 0.5% Tween20 in PBS).The primary antibodies were incubated with samples overnight at 37 °C, and the secondary antibodies were incubated for 3 h at 37 °C. The cells were washed 5 times with PBS. Finally, Hoechst nuclear stain (1 µg/µl) was added to the cells and incubated for 15 min at 37 °C. The fluorescent signal from the cells was observed with a confocal microscope (20×).

The fluorescent signals were detected in 3 colours, including blue (Hoechst, 510–540 nm), red (Cy3, 560–570 nm) and green (FITC, 500–520 nm), using a motorized fluorescence microscope type IX83 (Olympus Co., Ltd., USA). The protocol for signal intensity detection and the number of positive cells was operated by CellSens imaging software (Olympus Co., Ltd., USA). Briefly, 12 fields of 20× objective lenses (4 columns × 3 rows) at the centre of each well were captured with specific exposure times as follows: Hoechst (25.5 ms), Cy3 (700 ms) and FITC (316 ms). The signals with 25–200 µm perimeter and 140–300 mean grey intensity values were counted and exported to apply the CancerScreen.exe program (Please contact author for program requirement), which was developed by Aporntewan C. The positive signal in each colour was identified by CancerScreen.exe based on 2 criteria, including the range of perimeter (25–150 µm) and mean grey intensity value in pixels/cell volume units (150–280). The positive cells showed red and green signals in the same position, and the negative cells showed only red signals. Then, the average fluorescence intensity of anti-STAU2 in each sample was calculated from the intensity of positive cells divided by the intensity of positive and negative cells. Moreover, the percentage of STAU2-positive cells was calculated by the number of positive cells divided by the total number of cells. Positive samples from cocultured PBMCs were applied as interassay variation adjustments.

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