Ly6c pe

Mice under indicated conditions were anesthetized, and kidneys were harvested and cut into pieces. Then kidney tissues were digested in a buffer (HBSS supplemented with 0.05% collagenase I and 2 mM CaCl₂) under 37 °C for 25 min. After digestion, the kidney tissues were filtered through a 70-μm nylon mesh. The cell suspension was centrifuged at 500g for 5 min, and the resulting cell suspension was treated with Fcγ receptor blocker (101320, BioLegend) for 10 min followed by incubating with the following fluorescent antibodies (all from BioLegend): CD45 BV421 (103134), CD11b FITC (101206), Ly6G APC/Cyanine7 (127624), Ly6C PE (128008), F4/80 APC (123116), 7AAD (420404), and CD38 PE/Cyanine7 (102717). Flow cytometry was performed on a FACSCanto II (BD Biosciences), and data were analyzed by FlowJo software 10.4.

0.25 × 10⁶ SCLL cells were engrafted into 6–8-week-old, female Balb/C mice and PB was collected for flow cytometry analysis as described previously [23 (link)]. All animal experiments were performed under an approved protocol from the Augusta University Institutional Animal Care and Use Committee. Flow antibodies used in this study include CD4-APC (Biolegend, #100,412), CD8α-PE/Cy7 (Biolegend, #100,722), Ly6C-PE (Biolegend, #128,008), CD11b-PerCP/Cy5.5 (Biolegend, #101,228), Ly6G-APC/Cy7 (Biolegend, #127,624), CD19-Violet 421 (Biolegend, #115,538), CD49b-PerCP/Cy5.5 (Biolegend, #108,916).

Monoclonal fluorophore-conjugated antibodies used for flow cytometry were obtained from BD Biosciences (CD11b-APC-Cy7, CD45-PeCy7, CD11c-BV421, CD274-APC, CD69-APC, CD44-PeCy7 or FITC, CD184-PE, CD3e-FITC, Ly-6c-PE, Ly-6G-APC) and BioLegend (CD8a-BV785, CD45-PerCP, and ENPP1-BV421). For immunohistochemistry, rabbit anti-Iba1 monoclonal antibody (Abcam # ab178846) was used at 1: 500 dilution. Rat anti-mouse LAMP1 monoclonal Ab was obtained from the Hybridoma Bank (Cat # 1D4B) and used at 1: 250 dilution. Mouse anti-CD68 monoclonal Ab (Abcam # ab955) was used at 1: 200 dilution. To identify amyloid plaques, anti-Aβ monoclonal 4G8 (Aβ 17–42) antibody (Signet Cat # 9220–02) was used at 1: 1000 dilution. Secondary anti-mouse, anti-goat and anti-rabbit antibodies (Jackson labs and Sigma-Aldrich) were used for immunohistochemistry. In immunofluorescence studies, Rhodamine-conjugated donkey anti-rat IgG (1: 500), Alexa-488-conjugated goat anti-rabbit (1: 500), and DyLight 405-conjugated donkey anti-mouse (1: 500) were used as secondary antibodies. Fluorescent-labeled Aβ₄₂ fibrils for immunofluorescence studies were prepared as described below and FITC filter was used to observe Aβ fluorescence.

Liver non-parenchymal cells were isolated as previously described (Melino et al., 2016 (link)). Briefly, tissue disaggregation was performed by finely chopping liver samples (∼1-2 g) in 10 ml digestion solution containing 1 mg/ml Collagenase IV (Worthington) and 20 μg/ml DNAse1 (Roche) and incubating at 37°C for 45 min on a rocking platform before mashing through a 70 μm filter (Falcon). The cell pellet was collected by centrifugation (400 g) and resuspended in an isotonic 30% Percoll solution to separate hepatocytes and non-parenchymal cells. Cells were stained for a panel of myeloid markers [F4/80-AF647 (1:150), Cd11b-BV510 (1:200), Ly6G-BV785 (1:200), MHCII-BV421 (1:200), Tim4-PE-Cy7 (1:300), Ly6C-PE (1:300) (Biolegend)] in buffer containing 2.4G2 supernatant to block Fc binding, washed and resuspended in buffer containing viability dye 7AAD (Life Technologies) for acquisition using a Cytoflex (Becton Dickinson). Live single cells were identified for phenotypic analysis by excluding doublets (FSC-A>FSC-H), 7AAD+ dead cells and debris. Single colour controls were used for compensation and unstained and fluorescence-minus-one controls were used to confirm gating. Data were analysed using FlowJo 10 (Tree Star). Cell counts were calculated by multiplying the frequency of the cell type of interest by the total mononuclear cell yield/g of disaggregated tissue.

Peritoneal fluid samples were stained with fluorochrome-conjugated anti-mouse monoclonal antibodies: CD11b-APC (101212, BioLegend, San Diego, CA, USA), CD3-PE (100308, BioLegend, San Diego, CA, USA), Ly6C-PE (128007, BioLegend, San Diego, CA, USA), and Ly6G-FITC (127605, BioLegend, San Diego, CA, USA). Samples were analyzed using the CytoFLEX flow cytometer (Beckman Coulter, California, CA, USA). The CD11b+ leukocytes were gated by Ly6C/Ly6G into neutrophils (Ly6C^med/Ly6G⁺) and monocytes (Ly6C^high/Ly6G⁻). T cells were gated by CD3.