Apc anti mouse cd45 antibody

Mice were sacrificed 24 h after LPS exposure. Lung tissues and peripheral blood mononuclear cells were collected. To obtain BAL fluid, lungs were washed three times with 1 ml PBS. The single-cell suspensions for flow cytometric analysis were pre-incubated with anti-CD16/CD32 antibody (Cat# 101301, from BioLegend, San Diego, CA, USA) to block Fc receptors, and then stained with APC anti-mouse CD45 antibody (Cat# 103137, from BioLegend) and FITC anti-mouse Ly6G antibody (Cat# 127605, from BioLegend) at 4 °C for 20 min. Then, total cell numbers of immune cells including neutrophils were determined through the Cytoflex Flow Cytometer (Beckman Coulter, CA, USA).

To prepare single-cell suspensions, tumor tissues were minced and digested with 2 mg/ml collagenase type I (Worthington Biochemical, USA) for 1 h at 37 °C followed by filtration with 200-mesh gauze. The acquired single-cell suspensions were incubated at RT for 15 min with fluorescence-labeled antibodies against CD45, CD3, CD4, CD8, IFN-γ. For intracellular cytokine staining, the Intracellular Fixation and Permeabilization kit (Invitrogen, USA) was used according to the manufacturer's instructions. After staining, single-cell suspensions were detected by flow cytometry. The fluorescence-labeled antibodies used above were: APC anti-mouse CD45 Antibody (1:500, Biolegend, 103111), Brilliant Violet 421 anti-mouse CD3 Antibody (1:500, Biolegend, 100227), FITC anti-mouse CD4 (1:500, Biolegend, 100406), PE anti-mouse CD8a (1:500, Biolegend, 100708), 7-AAD Viability Staining Solution (1:500, BD, 559925).

A portion of the cell suspension was centrifuged at 300 x g for 5 min. 100 µL of wash solution was used to resuspend the cell precipitate, 2 µL of APC anti-mouse CD45 antibody (BioLegend, USA) was added for every 1× 10 ⁶ cells according to the total number of cells, and the mixture was incubated at 4 °C and protected from light for a total incubation time of 30 min. At 15 min of incubation time, 1 µL of calcein AM (BD, USA) was added for every 100 µL of cell suspension. After incubation, the cells were washed twice by centrifugation at 300 x g for 5 min; the supernatant was discarded, and the cells were resuspended and mixed with the appropriate amount of washing solution. A small amount of the suspension was stained with AO/PI, and the number of cells sorted, the cell viability and the clumping rate were recorded; the cells were centrifuged at 300 x g for 5 min and resuspended in sample buffer for BD quality control.