Anti cd11b fitc

Confluent monolayers of astrocytes on 12-well plate were digested with 0.25% trypsin, and the cell suspension was collected for flow cytometric analysis. Briefly, after the cells were washed with cold PBS, they were suspended in 0.2% bovine serum albumin (Biosharp) in PBS. The astrocytes were then stained with APC-anti-ACSA-2 (MACS), FITC-anti-CD11b (BD), PE-anti-CXCR3 (BD), or isotype IgG for 25 min at 4°C. The cells were washed twice with PBS and fixed with 1% paraformaldehyde. The populations of astrocytes were detected using a FACSCalibur (BD), and the results were analyzed using CellQUEST Pro software (BD).

The fluorescence-labeled monoclonal antibodies were as follows: FITC-anti-CD3, PerCp-Cy5.5-anti-CD8, APC-Cy7-anti-CD11b, PerCp-Cy5.5-anti-F4/80, APC-anti-CD4, and PE-Cy7-anti-CD45 were purchased from Biolegend (San Diego, CA, USA); FITC-anti-CD11b was from BD Biosciences (San Jose, CA, USA); and PerCp-Cy5.5-anti-F4/80 was from eBioscience (San Diego, CA, USA). MNCs were stained with the indicated antibodies as previously described^{37 (link)}. Data were collected on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo 7.6.1 (Tree Star, Ashland, OR).

To assess immune cells, cells were washed with PBS/0.5% BSA. Staining was performed with PE anti-CD11b (BD Pharmingen) and APC anti-CD45 (BD Pharmingen), or FITC anti-CD11b (1:100; BD Pharmingen), PE anti-CCR2 (1:20; R&D Systems) and APC anti-CD45 (1:200). Isotype control rat IgG2b (R&D Systems) was used for CCR2-antibody. Subsequently, cells were incubated for 20 min on ice. DAPI was added to detect dead cells.
For other surface molecules, cells were fixed. Here, the cell suspension was washed with PBS, fixed for 20 min with 2% PFA, and washed with PBS/0.5% BSA. Cells were permeabilized with 0.5% saponin (exception MHCII, PBS/0.5% BSA) and staining was performed in 0.5% saponin (MHCII only 0.5% BSA/PBS). Antibodies PE anti-CD11b (1:200) and APC anti-CD45 (1:200) were used in combination with the respective specific antibodies coupled to FITC: anti-I-Ab (1:50), anti-CD80 (1:100), anti-CD86 (1:100) as well as isotype controls rat IgG2a, Armenian hamster IgG and mouse IgG2a (BioLegend, San Diego, CA, USA). Cells were incubated for 20 min at room temperature. Cells were washed with 0.5% saponin and analyzed by flow cytometry.
All samples were analyzed with BD FACS Canto II (BD Pharmingen) and evaluated with FlowJo software (Ashland, OR, USA).

The estimated percent of glia in mixed glial, microglial, and astrocyte cultures were determined by immunophenotying using direct labeling with anti-GLAST-PE (Miltenyi Biotec, San Diego, CA), anti-Cd11b-FITC (BD Biosciences), anti-CD11b-PE (Stemcell Technologies), and anti-GLAST-488 (Novus Biologicals, Littleton, CO) followed by flow cytometric analysis. Cells were counted using a Bio-Rad TC10 automated cell counter, and 1 × 10⁶ cells/mL were resuspended in 100 μL of incubation buffer (PBS with 0.05% bovine serum albumin). Mixed glial cultures were labeled using the mouse anti-GLAST-PE (20 μg/mL) and mouse anti-Cd11b-FITC (10 μg/mL) at room temperature for 1 h. Microglia cultures were incubated with CD11b-PE according to manufacturer instructions while astrocyte cultures were incubated with rabbit polyclonal anti-GLAST-488 (10 μg/mL) at room temperature for 1 h. After labeling, the cells were washed twice in incubation buffer and resuspended at a final volume of 500 μL of PBS and stored at 37 °C until analysis. Flow cytometry was performed on a Beckman Coulter CyAn ADP flow cytometer operated with Summit software for data collection at Colorado State University’s Flow Cytometry Core Facility. All further data analysis was done utilizing FlowJo software (version 10.1; FlowJo, Ashland, OR).