Flow cytometry
For
surface staining, mouse-derived splenocytes were labeled for 30 min with
directly labeled antibodies against cell surface antigens. For
intracellular cytokine staining, cells were stimulated with 0.1 mg/ml
foponol-1-myristate-37-acetate (PMA) plus 11 ng/ ml ionomycin for 4 h at
37°C (all from Enzo Life Sciences, Farmingdale, NY, USA), followed by
fixation and permeabilization with BD Cytofix kits (BD Biosciences. the
USA) to fix and permeabilize the cells, followed by staining with
corresponding antibodies. To stain intranuclear factor Foxp3, membranes
were broken for 30 min using a fixation/permeability kit (Invitrogen,
USA) and then stained with APC-anti-Foxp3. To assess the cell viability,
cells were directly stained with the reactive dye eFluor 506 for 20 min.
For cell proliferation and apoptosis, intranuclear staining with the
proliferation marker Ki67 was performed for 30 minutes, and cell
apoptosis was assessed with Annexin V-PE/7-AAD Apoptosis Detection Kit
(Vazyme Biotech, Nanjing). All antibodies used for flow cytometry
detection were listed in Table S2. The assays were performed on a BD
FACSAria III flow cytometer (BD Biosciences), and the data were analyzed
using FlowJo X software.
Laboratory test
Proteinuria was quantitatively detected by a urine protein detection kit
(Nanjing Jiancheng, China). Serum and urine creatinine levels were
measured by a creatinine detection kit (Nanjing Jiancheng, China). Urea
nitrogen levels were measured using a urea nitrogen kit (Nanjing
Jiancheng, China). Immunoglobulin G (IgG) and antibody levels were
measured using mouse IgG (Multi Sciences, China) and anti-nuclear
antibody (ANA), anti-double strand DNA (dsDNA) (Aifang Bioscience,
China) ELISA kits.
Renal histology and
immunofluorescence
Renal tissue samples fixed in formaldehyde fixative were embedded in
paraffin and cut into 10μm sections. Hematoxylin-eosin (H&E)
staining was used to observe the
changes in glomeruli under the microscope refer to the previous
description[11[] Chen, Weiwei et al.
“Lipocalin-2 Exacerbates Lupus Nephritis by Promoting Th1 Cell
Differentiation.” Journal of the American Society of Nephrology :
JASN vol. 31,10 (2020): 2263-2277. doi:10.1681/ASN.2019090937].
Immunofluorescence staining of paraffin-fixed kidney sections with
Rhodamine conjugated anti-IgG (Jackson Immuno Research, USA) and FITC
conjugated anti-complement 3 (C3) (Abcam, UK) antibodies was performed
to observe IgG and C3 deposition under a fluorescence microscope, as we
described previously[22[] Zhang, Zhuoya
et al. “Mesenchymal stem cells prevent podocyte injury in lupus-prone
B6.MRL-Faslpr mice via polarizing macrophage into an anti-inflammatory
phenotype.” Nephrology, dialysis, transplantation : official
publication of the European Dialysis and Transplant Association -
European Renal Association vol. 34,4 (2019): 597-605.
doi:10.1093/ndt/gfy195].