Igg2a pe

At the end of the experiment, spleens were isolated from ApoE KO mice and placed in a chilled RPMI-1640 medium (Gibco, Carlsbad, CA, USA), mashed through a 70 µm nylon cell filter (Corning, Corning, NY, USA), and centrifuged at 400× g for 5 min. The obtained splenocytes were stained with relevant antibodies. The following antibodies were used: CD4-PE (553048, PE Rat Anti-Mouse CD4, BD Biosciences, Franklin Lakes, NJ, USA), CD8-PE (553032, PE Rat Anti-Mouse CD8a, BD Biosciences, USA), and FoxP3-Alexa Fluor 488 (560403, Alexa Fluor 488 Rat anti-Mouse Foxp3, BD Biosciences, USA). The following isotype match antibodies were used: IgG2a-PE (551799, PE Rat IgG2a, k Isotype Control, BD Biosciences, USA), and IgG2b-FITC (556923, FITC Rat IgG2b, k Isotype Control, BD Biosciences, USA). A flow cytometry analysis was performed on a Beckman Coulter Flow Cytometer (BD Biosciences, USA). Obtained data were analyzed using FlowJo software (FlowJo LLC, Franklin Lakes, NJ, USA).

CD14+ cells present in isolated PBMC were determined using anti-human CD14-PE (clone M5E2, BD Biosciences, Franklin Lakes, NJ) and an isotype matched negative control, IgG_2a-PE (BD Biosciences). PBMC were stained for 30 minutes at 4°C, washed with 1% BSA in PBS and fixed with 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Immunopositive cells were analyzed by acquisition of 10,000 events on a FACS Canto II flow cytometer and analyzed using FlowJo (Treestar, Ashland OR). To analyze CD4 or CCR5 protein expression on the macrophage surface, MDM were detached from culture dishes with TrypLE Select (10X) for 30 minutes at 37°C, followed by gentle agitation and scraping. 1–2×10⁵ MDM were stained for CCR5 or CD4 using anti-human CCR5-APC/Cy7 and CD4-FITC, with the isotype-matched negative controls, IgG₁-APC/Cy7 or IgG₁-FITC (BD Biosciences). Antibodies were titrated to determine the optimal concentration for staining PBMC and MDM. After subtracting out the background fluorescence from the isotype matched control, the mean fluorescence intensity (MFI) for each antigen was determined.

For flow cytometry, the following antibodies were purchased from BD Biosciences: CD25-APC (PC61), CD3-FITC (17A2), CD3-APC-Cy7, CD4-PE (L3T4), CD8a-FITC (LY-2, 53-6.7), CD8a-APC (53-6.7), B220-PE (CD45RA-14.8), Annexin V-APC, CD45-V500, CD14-V450, IgG_2a-PE rat isotype (R35–95), IgG_2a-APC rat isotype), IgG_2a-FITC, IgG_2a-V500, IgG_2a-V450 rat isotype. CD44-PE (IM7) was purchased from Biolegend, San Diego, CA, USA. Thymocytes, splenocytes and whole blood cells (1 × 10⁶ cells) were incubated with fluorochrome-conjugated antibodies (0.5 μg) for 30 minutes on ice, avoiding light. After two PBS washes, cells were analysed by flow cytometry using BD FACSAria (BD Biosciences) flow cytometer and BD FACSDiva software (BD Biosciences), as previously described76 (link). Splenic B-cells (B220+), splenic T-cells (CD3+), splenic CD4+ and CD8+ cells were sorted by antigenic criteria using BD FACSAria flow cytometer/cell sorter, with a purity grade of 99%.

In accordance with the literature, the most relevant markers have been selected for degranulation analysis [17 (link)]. Degranulation was determined by measuring the expression of CD markers characteristic for azurophil granules (CD63-PE), specific granules (CD15-FITC, CD66b-FITC), gelatinase granules (CD11b-PE), and secretory vesicles (CD13-APC, CD14-APC, CD18-FITC, and CD45-APC) at the plasma membrane by flow cytometry (all antibodies are from BD Biosciences except CD14-APC from Immunotools).
IgG1-FITC, IgG2a-PE (BD Biosciences), and IgG1-APC antibodies (Immunotools) were used as negative isotype controls to place the cells in the first decade of any plot, whereas CD45-FITC, CD45-PE, or CD45-APC (BD Biosciences) single dye staining was used to set compensations. Data analysis was performed by measuring the mean fluorescence intensity (MFI) for each CD marker with BD FACSDiva software (BD Biosciences) on the gated population of granulocytes (FSC-A versus SSC-A), single (SSC-A versus SSC-H), and living cells (negative cells for Sytox Blue staining (Invitrogen)). In total, 10,000 events were recorded per staining. The relative translocation of CD markers to the plasma membrane for each granule was determined by calculating the ratio between MFI of LPS-stimulated cells and nonstimulated control from the same time point.