Cell culture and viability analysis
The Raw264.7 cell line (Cell Resource Center of the Shanghai Institutes
for Biological Sciences (TIB-71), China) is a normal mouse macrophage
cell line. The Raw264.7 cell line is a type of adherent cell that was
cultured in DMEM (Shanghai Reith Biotechnology Co., Ltd., China)
supplemented with 10% FBS (Gibco, 10099-141, USA) in an incubator at 37
°C with 5% CO2. Cell viability was measured by the CCK-8 assay,
live-dead cell staining and cell apoptosis assay. For the CCK-8 assay,
Raw264.7 cells were seeded overnight in 96-well plates at a density of 5
× 104 cells/well. Then, the medium was changed to
medium with 10% FBS containing different percentages of P.g(109 CFU) culture medium (from 1% (1%-P.g ) to
10% (10%-P.g ) by volume) and incubated for 24 h (the total
volume of this mixture was 3 ml, and the control group was supplemented
with cell culture medium containing fresh BHI medium). CCK-8 solution
(10 µl/well) (Shanghai Reith Biotechnology Co., Ltd., China) was added
and incubated for 90 min at room temperature, and then the 96-well plate
was placed in a microplate spectrophotometer to measure the absorption
value at 450 nm. For live-dead cell staining, after the Raw264.7 cells
were seeded in 6-well plates at a density of 1 × 106cells/well overnight, they were cultured with 5% P.g for 24 h.
Cell viability was determined using the Live-Dead Cell Staining Kit
(Vazyme Biotech Co., Ltd., China) in accordance with the manufacturer’s
instructions. Images were captured with a Carl Zeiss LSM710 fluorescence
microscope. The SV40-MES-13 cell line (Cell Resource Center of the
Shanghai Institutes for Biological Sciences (CRL-1927), China) is a
normal mouse glomerular mesangial cell line. The SV40-MES-13 cell line
was cultured in DMEM/F12 medium (Shanghai Reith Biotechnology Co., Ltd.,
China) supplemented with 10% FBS (Gibco, 10099-141, USA) in an
incubator at 37 °C with 5% CO2. Cell viability was measured as
previously described. For the CCK-8 assay, SV40-MES-13 cells were seeded
in 96-well plates at a density of 5 × 104 cells/well
overnight. Then, the cell culture medium was changed to medium
containing 10% FBS containing 50% (by volume) and the supernatant of
RAW264.7 cells stimulated with 5% P.g (macrophage-conditioned medium,
MΦCM) for 24 h (the total volume was 3 ml, and the control group was
supplemented with the supernatant of RAW264.7 cells without P.gstimulation). CCK-8 solution (10 µl/well) (Shanghai Reith Biotechnology
Co., Ltd., China) was used to assess the viability of the cells. After
being incubated for 90 min at room temperature, the 96-well plate was
placed in a microplate spectrophotometer to measure the absorption value
at 450 nm. After SV40-MES-13 cells were seeded in 6-well plates at a
density of 1 × 106 cells/well overnight, they were
cultured with MΦCM for 24 h. For live-dead cell staining, viability was
determined using the Live/Dead Cell Staining Kit (Vazyme Biotech Co.,
Ltd., China) in accordance with the manufacturer’s instructions. Images
were captured with a Carl Zeiss LSM710 fluorescence microscope.