Dulbecco s pbs

Cryopreserved PBMCs were thawed as previously described (37 (link)), and resuspended at 5 × 10⁶ cells/ml in PBS. Cells were incubated with 10% FCS at 4°C for 10 min prior to staining for 30 min with PerCP-Cy5.5 conjugated anti-CD3 (clone OKT3 from eBiosciences, San Diego, CA, USA). Cells were permeabilized with permeabilization buffer [made up of 0.1% NaAzide and 0.1% saponin in Dulbecco’s PBS (Lonza, Verviers, Belgium)], and incubated with FITC conjugated anti-CD3ζ antibody (clone 6B10.2 from BioLegend, San Diego, CA, USA). At least 50,000 live events were acquired on an LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis was performed using FlowJo software (TreeStar, USA) and mean fluorescence intensity (MFI) was calculated for CD3 and CD3ζ. CD3ζ expression on T cells was determined according to expression levels on CD3 positive cells, and normalized by subtracting the MFI of CD3 in the CD3 negative population.

The blood and the regions of interest in the ipsilateral side of the brain (hippocampus, amygdala, and cortex) were taken. Five animals (all the conditions) were processed per day with a total of 20 samples at the time. Mice were anesthetized with isoflurane, and blood samples were taken in EDTA tubes immediately before transcardial perfusion with ice-cold 0.1 M heparinized-PBS. The brain regions of interest were dissected out on ice and digested at 37 °C for 15 min with collagenase D (400 units/mL, Roche) in Dulbecco’s PBS (Lonza, Basel, Switzerland), each containing 50 μg/mL of DNase I (Sigma-Aldrich). The tissue was then mechanically dissociated with a glass Pasteur pipette, filtered through a 70-μm nylon cell strainer, and centrifuged at 950 rpm for 15 min. A 25% Percoll (Sigma-Aldrich) column was used to remove cell debris and myelin, followed by centrifugation at 1700 rpm for 10 min. 50 µl-blood sample was mixed with 1 × Red Blood Lysis Buffer (Roche) and incubated in rotation for 15 min at room temperature (RT). Samples were then centrifuged at 3500 rpm for 5 min at RT. The supernatant was discarded, and cells were washed and resuspended in 1 mL of cytometer buffer [0.5% bovine serum albumin (Sigma-Aldrich), 5 mM EDTA (Millipore, Burlington, MA) in PBS]. The cells were resuspended in 100 µl of cytometer buffer and stained.

Cells were stained with specific mAb or appropriate isotype controls for 30 min at 4°C in FACS buffer (Dulbecco's PBS [Lonza] containing 2% FCS [PAA/GE Healthcare]), washed twice, and finally resuspended in cold FACS buffer containing 0.1 μg/mL propidium iodide (PI) (Carl Roth, Karlsruhe, Germany). Stained cells were immediately analyzed with a FACScan cell analyzer (BD Biosciences). Cell debris and dead cells were excluded from the analyses by gating on proper forward and sideward light scatter and on PI negative cells. Also, percentages of PI positive cells were calculated using this gating strategy. A minimum of 10⁴ living cells was measured for each sample and results were analyzed using FCS Express 4 Flow Cytometry Software (De Novo Software, CA, US). The following monoclonal antibodies (all from BD Biosciences) were used to determine the phenotype of DCs: PE-labeled mouse anti-human CD25 (M-A251), CD80 (L307.4), CD83 (HB15e), CD86 (IT2.2), HLA-ABC (G46-2.6), and HLA-DR (G46.6). Isotype mAb controls (all from BD Biosciences) used but not shown were IgG1-PE (MOPC-21), IgG2a-PE (G155-178), and IgG2b-PE (27-35).

Adult mice were anesthetized with i.p. ketamine/xylazine and perfused transcardially with ice-cold phosphate-buffered saline (PBS, Lonza, Verviers, Belgium). The striatum and midbrain were dissected on ice and digested with a collagenase D mix (400 units/mL, Roche, Mannheim, Germany) or a papain mix (2 mg/mL, Roche, Mannheim, Germany) containing DNase-I (50 µg/mL, Sigma-Aldrich) in Dulbecco’s PBS (DPBS, Lonza, Walkersville, MO, USA). The tissue was digested at 37 °C under rotation, for 15 min for collagenase D or 30 min for papain. To stop the reaction, the samples were transferred at 4 °C and 10 µL of EDTA 500 mM (Invitrogen, Grand Island, NY, USA) were added. Finally, the brain tissue was dissociated mechanically with a glass pipette, filtered through a 70 μm nylon cell strainer humidified with cold DPBS and centrifuged at 300 g for 15 min at 4 °C. The pellet was resuspended with 25% Percoll (GE Healthcare, Uppsala, Sweden) and centrifuged at 1000 g for 10 min at RT to remove cell debris and myelin. The fat-containing white layer was carefully removed and the cell pellet was resuspended in the appropriate buffer for RNA sequencing (RNA-seq) or flow cytometry.

The blood and the ipsilateral cortex and hippocampus were taken 7 days after CCI or sham surgery. Mice were anesthetized with isoflurane, and blood samples were taken in EDTA tubes immediately before transcardial perfusion with ice-cold 0.1 M heparinized-PBS. The brain regions of interest were dissected out on ice and digested at 37 °C for 15 min with collagenase D (400 units/mL, Roche) in Dulbecco’s PBS (Lonza, Basel, Switzerland), each containing 50 μg/mL of DNase I (Sigma-Aldrich). The tissue was then mechanically dissociated with a glass Pasteur pipette, filtered through a 70-μm nylon cell strainer, and centrifuged at 950 rpm for 15 min. A 25% Percoll (Sigma-Aldrich) column was used to remove cell debris and myelin, followed by centrifugation at 1700 rpm for 10 min. 50 µl-blood sample was mixed with 1 × Red Blood Lysis Buffer (Roche) and incubated in rotation for 15 min at room temperature (RT). Samples were then centrifuged at 3500 rpm for 5 min at RT. The supernatant was discarded, and cells were washed and resuspended in 1 mL of cytometer buffer [0.5% bovine serum albumin (Sigma-Aldrich), 5 mM EDTA (Millipore, Burlington, MA) in PBS]. The cells were resuspended in 100 µl of cytometer buffer and stained.