Cd11c pe cy7

Single cells isolated from spleen and intestinal lymph node were stained for FACS analysis as described previously (16 (link)). Lymphocytes were stained using the Zombie Violet™ Fixable Viability Kit (BioLegend Biotechnology Co., LTD, China) and were stained for surface markers including CD3-APC-Cy7 and CD8-APC (Thermo Fisher Scientific Co., LTD., USA); cells were then fixed and permeabilized by the Intracellular Fixation and Permeabilization Buffer Set (Thermo Fisher Scientific Co., LTD., USA) and stained for intracellular expression markers such as IFN-γ-PerCP and IL-4-PE (Thermo Fisher Scientific Co., LTD., USA). DCs were stained for surface markers including CD11c+-PE-Cy7, CD80-APC, CD86-FITC, and MHC-II-PE (Thermo Fisher Scientific Co., LTD., USA). Data were acquired with BD FACSVerse (Becton Dickinson Co., LTD., Canada) and analyzed with BD FACSuite (Becton Dickinson Co., LTD., Canada).

Following Fc block (eBiosciences, 93) cells were stained with the following antibodies: CD11a-PE (BioLegend, 2D7), CD18-FITC (BD Biosciences, C71/16) CD11c-PE-Cy7 (eBioscience, N418), CD11b-APC (BioLegend, M1/70), CD80-APC (eBioscience, 16-10A1), CCR7-PE (BioLegend, 4B12), CD40-PE (BioLegend, 3/23), CD86-FITC (BD Biosciences, GL1). FITC-conjugated antibodies were used at 1:100 dilution, all other antibodies were used at 1:200 dilution. Data were acquired using a LSRForetessa (BD) and analyzed using FlowJo software (TreeStar, USA).

The cells were phenotypically characterized by flow cytometry using the following conjugated antibodies (Abs): mouse anti human-HLA-ABC-FITC, CD80-FITC, CD83-FITC, CD86-FITC, CCR7-FITC, and CD11c-PE-Cy7 (eBioscience, San Diego, CA, USA). Briefly, cells were gently removed from the culture plates using cell scrapers. Then, the cells were centrifuged at 1000 rpm for 5 min at 4 °C, washed with PBS and incubated with Abs for 30 min. After being washed twice with PBS, samples were acquired on a FACSVerse (BD Biosciences, Hershey, PA, USA) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Cell viability was verified through the viability dye LIVE/DEAD (Thermo Fisher Scientific Inc., Waltham, MA, USA). All the analyses were made in the CD11c+ cell population of each condition and sample.

Mononuclear cells were isolated from brain and spinal cord of CX3CR1-WT, CX3CR1-KO, and hCX3CR1^I249/M280 mice by homogenization of CNS tissues as previously described (Pino and Cardona, 2011 (link)). Cellular suspensions were blocked using anti-mouse CD16/CD32 (clone 2.4G2, BD Pharmingen) followed by incubation for 30 min with a mix of fluorochrome-conjugated anti-mouse antibodies as follows. Antibody cocktail for mononuclear myeloid cells and activation markers: CD45 APC-Cy7 (clone 30-F11, BioLegend), CD11b PE-CF594 (clone M1/70, BD Biosciences), CD11c PE-Cy7 (clone N418, eBioscience), I-A/I-E BV421 (MHC-II clone M5/114.15.2, BD Biosciences), CD86 PerCP/Cy5.5 (clone GL-1, BioLegend), Ly6C AF647 (clone ER-MP20, Serotec), Ly6G PE (clone 1A8, BD Pharmingen). T cells were characterized using the following antibody mix: CD45 eFluor 450 (clone 30-F11, eBioscience), CD3e PE-Cy7 (clone 145-2C11, BioLegend, CD8a APC (clone 53-6.7, eBioscience), CD4 APC-Cy7 (clone RM4-5, BioLegend), CD44 PerCP (clone IM7, eBioscience). Samples were acquired on an LSRII (BD Biosciences) at the Cell Analysis Core, UTSA. Data analysis was performed using FlowJo v10. For cell sorting, brain cell suspensions (pooled 3 mice per sample) were stained with antibodies against CD45 and CD11b and after gating for single cells the CD45^LoCD11b⁺ population was separated in a FACS ARIA-II (BD Biosciences).

Animals were sacrificed and the spleens and kidneys were collected for flow cytometry analysis. The monoclonal antibodies used for splenic flow cytometry were CD4-PE, CD3-PE-cy7, Foxp3-FITC, CD69-percp-cy7, B220-PE-cy7, CD21-FITC, CD23-PE, CD11c-PE-cy7, CD11b-APC, F4/80-PE, CD86-FITC, and F4/80-PErCP. The monoclonal antibodies used for renal flow cytometry were CD4-PE, CD3-Percp, Foxp3-FITC, CD45-APC-cy7, CD11b-FITC, CD11c-PE-cy7, F4/80-PE, and Gr-1-Percp (eBioscience, Hanover Park, IL, USA). Cell counting was performed by using a Cellometer^® automated cell counting system (Sigma-Aldrich, St Louis, MO, USA) for absolute cell numbers. The Novocyte flow cytometer system (ACEA Bioscience Inc., San Diego, CA, USA) was used for flow cytometry, and analysis was performed as described [11 (link)]. Data were analyzed using Novocyte software (ACEA Bioscience Inc.). At least 200,000 events were acquired for each analysis.

A nonparenchymal cell fraction from whole liver extracts was isolated upon collagenase and mechanical digestion followed by Percoll (GE Healthcare Life Sciences) gradient centrifugation as previously described [5 (link)]. In parallel, blood samples were collected in EDTA-containing tubes and treated with red blood cell lysis buffer (PharmLyse, BD Biosciences, Germany). Upon removal of red bodies and centrifugation, immune cells were incubated with fluorochrome-conjugated antibodies and characterized according to two different panels, a myeloid panel: CD45-BV510 (103138, BioLegend), 7AAD-PE-Cy5-YG (420404, BioLegend), CD11b-BV711 (101242, BioLegend), F4/80-APC (17-4801-82, eBiosciences), MHC2-Alexa700 (107622, BioLegend), CD11c-PE-Cy7 (25-0114-81, eBiosciences), and Ly6G-FITC (551460, BD Pharmingen) and a lymphoid panel: CD45-BV510 (103138, BioLegend), 7AAD-PE-Cy5-YG (420404, BioLegend), CD3-PE-Cy7 (25-0031-82, eBiosciences), CD4-FITC (11-0041-85, eBiosciences), CD8-PerCpCy5.5 (126610, BioLegend), and NK1.1-BV711 (108745, BioLegend). Labeled cells were then subjected to flow cytometry using a BD Canto II (BD Biosciences) and relative cell numbers were analyzed using FlowJo software (Tree Star).