Lsr 2 flow

Cells were stained with anti-CD40, anti-ICAM-1 (eBiosciences, San Diego, CA, USA), or isotype control mAbs. Cells were analyzed on an LSR II flow cytometer (Becton Dickinson, San Jose, CA, USA). FlowJo software version 10.10 (Tree Star Inc., Ashland, OR, USA) was used for analysis.

Following MSA treatment, adherent cells were washed and labeled with 5 μM BODIPY C-11 in DMEM lacking serum and antibiotics for 15 min at 37 °C. Following labeling, cultures were collected by trypsinization, washed, resuspended in cold PBS and filtered. Samples were read on a Becton-Dickinson LSR II flow cytometer using channels for Texas Red (reduced dye) and fluorescein isothiocyanate (FITC, oxidized dye) simultaneously at The University of Iowa Flow Cytometry Core. Populations were gated and analyzed with FlowJo software (version 7.6.5), and ratios of oxidized:reduced dye were calculated.

Activated CD4 and CD8 T cells were identified using the following staining panel: brilliant™ violet (BV) 570-conjugated anti-CD3 (clone: UCHT1), phycoerythrin (PE) Cyanine5.5-conjugated anti-CD4 (clone: MHCD0418), BV605-conjugated anti-CD8 (clone: RPA-T8), CD38 BV711 (clone: HIT2) and BV785-conjugated anti-HLADR (clone: UCHT1). All conjugated antibodies were purchased from Biolegend (San Diego, CA, USA) except for the PE-Cy5.5 conjugated anti-CD4, which was purchased from Invitrogen. CD4+ and CD8+ T cells were gated within single, viable CD3+ lymphocytes. Appropriate controls including fluorescence minus one controls, were done. Data analysis was performed with FlowJo V10 software (Tree Star, Ashland, OR, USA). Flow Staining was acquired within 24 hours of staining on a LSRII Flow (Becton Dickinson Biosciences, San Jose, CA, USA).

Human albumin (cat# E80–129 Bethyl Laboratories), human IgM (cat# 109–035–043 Jackson ImmunoResearch), and human IgG (cat# 109–035–008 Jackson ImmunoResearch) levels were quantified in the plasma by ELISA according to the manufacturer’s instructions and previously described protocols [16 (link)]. Mononuclear cells from blood, spleen, bone marrow, thymus and lymph nodes were isolated as previously described [16 (link)]. Liver leukocytes were isolated after eliminating circulating cells by perfusing the liver with PBS in situ through the vena cava as previously described [28 (link)]. To test the functionality of T cells, splenocytes were cultured in IMDM with 10% fetal calf serum, 10 IU/ml hIL-2 (Chiron), 100 ng/ml hIL-7 (Miltenyi), 4x10⁵ Dynabeads CD3/CD28 (Life technologies) for 9 days then stimulated for 4 hours with 50 ng/ml PMA and 1 microg/ml ionomycin for 4 hours prior to intracellular cytokine staining using the BD cytofix/ cytoperm method (BD biosciences). Flow cytometry was performed with directly conjugated antibodies according to standard techniques using a Fortessa and LSRII flow cytometers (Becton Dickinson). LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) and a 405 nm excitation were used to exclude dead cells. The list of anti-human antibodies used in flow cytometry is detailed in S1 Table. Analysis was performed with Flowjo Version 8.8 (TreeStar).