Alexa fluor 488 affinipure goat anti rabbit igg

RAW264.7 cells (5 × 10⁵ cells) were seeded and incubated with LPS or LPS in the presence of LDL. The cells were treated with DS-Ce6 (equiv. 5 μM Ce6) for 1 h, irradiated using a 670 nm laser (50 mW, irradiated area 3.8 cm²) for 10 s, and further incubated for 15 min, 30 min, and 1 h. Cells without laser irradiation were used as a control. The cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature, blocked with 1% BSA for 30 min, and incubated with LC3 antibody (1:200; Novus Biologicals) for 1 h at room temperature, followed by incubation with Alexa Fluor 594 AffiniPure goat anti-rabbit IgG (1:100) in the dark. After washing three times with PBS for 5 min each in the dark, the cells were incubated with p62 antibody (1:200; Abcam) for 1 h at room temperature and then with Alexa Fluor 488 AffiniPure goat anti-rabbit IgG (1:100) in the dark. After washing with PBS, the cells were mounted using a fluorescence mounting medium with DAPI. Fluorescence was observed using a model LSM 780 Meta NLO confocal microscope (Carl Zeiss). The LC3 puncta per cell of at least 20 cells in each experimental arm was counted by a blinded observer (n = 5).

Cells were trypsinized and collected together with medium after being exposed to CS or fresh air. The cells were centrifuged (660× g for 5 min) and the medium was removed. The cells were fixed in Paraformaldehyde (4% in DDW) for 10 min then washed with PBS without Ca⁺² and Mg⁺². The cells were resuspended in 800 μL of PBS containing 0.2% Tween (PBS-T) for 10 mins on ice. The cells were washed again with PBS and resuspended in 100 μL of PBS-T containing 3% BSA and anti-TSPO or anti-cAMP antibodies (1:100) (Abcam, Cambridge, UK). The resuspended samples were incubated overnight at 4 °C. On the next day, the cells were washed and resuspended in 100 μL of PBS-T containing 3% BSA and Alexa Fluor 488 AffiniPure Goat Anti-Rabbit IgG (1:1.000) (Abcam, Cambridge, UK). Mean fluorescence intensity (MFI) was measured using fluorescence assisted cell sorting (FACS) with a CyAN ADP FACS machine (Beckman Coulter, Brea, CA, USA). The data were analyzed using FlowJo software (10th version, FlowJo LLC, Ashland, OR, USA).