Siglec h

Isolated lymphocytes were stained for cell viability, surface, and intracellular Ags as previously described [18 (link)]. Briefly, cells were stained for viability using a LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation (ThermoFisher). Lymphocytes were washed with DPBS (Gibco) plus 10% FBS (Atlanta Biologicals), and then labeled with mAbs specific for MHC class II (I-A^k; Invitrogen), TCR-β, CD4, CD25, CD44, CD11c, CD8, CD86, OX40L, Siglec-H (eBioscience, San Diego, CA), CD62L (BD-Pharmingen, San Diego, CA), CCR6, CCR7, CXCR3, CXCR4, CD11b, CD40, CD80, and TGF-β (Biolegend, San Diego, CA). Cells were then fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (BioLegend), and labeled with mAbs specific for IFN-γ (BD-Pharmingen), IDO, IL-10, Foxp3, IL-1β (eBioscience), TNF-α (Biolegend), IFN-α (PBL Assay Science, Piscataway, NJ), or IFN-β (Biolgend) (Supplemental Table 1). Fluorescence was acquired on a BD Fortessa flow cytometer using BD FACSDiva software. All data collected from individual samples were analyzed using FlowJo software (Tree Star, Ashland, OR).

Cells were stained with antibodies specific for B220, CD19, CD21, CD23, IgM, CD11c, PDCA1, Siglec H, CD86, CD95, GL7, CD4, CD8, F4/80, and Ly6G (eBioscience), CD43, CD80, I-A/I-E, IFN-γ, and IgD (Biolegend). IFN-γ Intracellular labeling was done following cell fixation with Cytofix/Cytoperm kit (PharMingen), according to manufacturer’s suggested protocol. Data was acquired on a LSRII flow cytometer (BD Biosciences). Data files were analyzed using FCS Express software (De Novo).

Flow cytometry was performed using a FACS Calibur (BD Biosciences, San Diego, CA, USA) and FlowJo (Version 9.7.6)(Tree Star, Hazel Green, WI, USA) were used for all analyses. Total spleen count was acquired for each mouse and used for calculation of total number of each splenic cell subset. Cells were incubated with unlabeled anti-CD16/32 antibodies to reduce nonspecific Fc receptor–dependent binding of fluorescence-labeled antibodies Fluorescently-labeled antibodies with the following specificities were used for spleen cell characterization: B220, CD3, CD4, CD8, CD11c, CD19, CD21/35, CD23, CD25, CD44, CD62L, CD69, CD86, CD93, CXCR5, GL7, IgD, IgM, MHC Class II (I-A^b), PD-1, PDCA1, and Siglec-H (all from e-Bioscience, San Diego, CA, USA).

Hematopoietic progenitors, erythroid precursors, and mature leukocytes were assessed from the bone marrow of infected and uninfected miR-22 KO animals and WT controls 6 days post infection. Flow cytometric markers used to analyze various populations were as follows: HSCs (Lin⁻ cKit⁺ CD150⁺ CD48⁻ CD34⁻), myeloid (GR1⁺), B cells (B220⁺), and T cells (CD4⁺ or CD8⁺), common myeloid progenitors (CMPs) (Lin⁻ cKit⁺ IL7ra⁻ CD34⁺ CD16/32⁻), granulocyte-monocyte progenitors (GMPs) (Lin⁻ cKit⁺ IL7ra⁻ CD34⁺ CD16/32⁺), megakaryocyte-erythroid progenitors (MEPs) (Lin⁻ cKit⁺ IL7ra⁻ CD34⁻ CD16/32⁻), megakaryocyte progenitors (Lin⁻ CD34⁺ CD41⁺), and stages in erythroid development (Lin⁻ CD71^+/− Ter119^+/−); plasmacytoid DCs (CD11c⁺ SiglecH⁺ B220⁺ CD11b⁻); conventional DCs (CD11c⁺ CD11b⁺ B220⁻); T cells (CD3⁺); B cells (CD19⁺); mature neutrophils (Gr1^hi CD11b⁺); immature neutrophils (Gr1^lo CD11b⁺).
Antibodies used included: CD45.2 (104), CD48 (HM48-1), CD71 (R17217), Ter119, CD117 (2B8), Sca1 (D7), CD4 (GK1.5), CD8 (53-6.7), Mac1/CD11b (M1/70), F4/80 (BM8), CD150 (MSHAD150), CD11c (N418), SiglecH (eBio440c), B220 (RA3-6B2), CD11b (M1/70), CD19 (eBio1D3), CD3 (145-2C11) obtained from eBiosciences. CD45.1 (A20) was obtained from BioLegend and Gr1 (RB6-8C5) was obtained from BD.