Lympholyte separation medium

Whole blood of a subset of individuals (5 AD, 5 MCI, and 5 HC) was collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson). Peripheral blood mononuclear cells (PBMC) were separated on lympholyte separation medium (Cedarlane, Hornby, Ontario, CA, USA) and washed twice in PBS at 1500 rpm for 10 min; viable leukocytes were determined using a TC20 Automated Cell Counter (Biorad, Hercules, CA, USA).

To differentiate bone marrow Mφs (BMMφs), we generated the L929 cell (European Collection of Cell Cultures) supernatant by plating 2.5 × 10⁵ cells into a T150 flask with 50 ml of D10 media and by incubating for 12 days (37°C, 5% CO₂). After 12 days, the supernatant was collected and sterile filtered.
BMMφs were derived following procedures that have been previously described (82 (link)). Bone marrow was flushed from the tibias and femurus of mice, and single-cell suspensions were created. Lympholyte separation medium (Cedarlane Laboratories) was used to isolate mononuclear cells, which were plated in a 60-mm petri dish with 6 ml of BMMφ differentiation media [D10 media with 10% (v/v) L-cell supernatant]. Cells were cultured overnight (37°C, 5% CO₂), and nonadherent cells were transferred onto non–tissue culture–treated 100-mm petri dishes (1-ml cells per petri dish) with 7 ml of BMMφ differentiation media. Cells were incubated for 6 days (37°C, 5% CO₂) with an additional supplementation of 5 ml of BMMφ differentiation media on day 4 to promote BMMφ differentiation. On day 7, ice-cold PBS was used to remove BMMφs from the dish. BMMφ cultures were 98% CD11b⁺, I-A^lo, and B7.2^lo.

Fifty ml of whole blood were collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson & Co., Rutherford, NJ, USA). Peripheral blood mononuclear cells (PBMC) were separated on lympholyte separation medium (Cedarlane, Hornby, Ontario, CA) and washed twice in PBS at 1500 RPM for 10 min; viable leukocytes were determined using a Scepter 2.0 Handheld Automated Cell Counter (Millipore, Billerica, MA).

Whole blood (10 ml) was collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson & Co.). Peripheral blood mononuclear cells (PBMCs) were separated on lympholyte separation medium (Cedarlane, Hornby, Ontario, CA, USA) and washed twice in PBS at 1,500 rpm for 10 min; viable leukocytes were determined with Bio-Rad TC20 Automated Cell Counter (Bio-Rad Laboratories, Hercules, CA, USA).

The Spi-B/POU2AF1 gene expression analysis in peripheral blood mononuclear cells (PBMCs) could be performed at baseline and after 24 months of therapy in 13 patients with RRMS and 10 age- and sex-matched HCs. PBMCs were isolated from peripheral blood by density gradient centrifugation (Lympholyte Separation Medium, Cedarlane, Hornby, Ontario, CA, USA) and stored at −80°C until use.
Total RNA was extracted from PBMCs (RNAeasy Mini extraction kit, Qiagen), quantified and reverse-transcribed to cDNA, as previously described (19 (link)). RNA quality and purity were assessed by the analysis of spectrophotometric of 260/280 (≥2.0) and 260/230 ratio (range of 2.0–2.2). Pre-designed and validated assays (Thermo Fisher Scientific) were used for relative quantification of the target genes (Spi-B and POU2AF1) and for two previously selected housekeeping genes (GAPDH, YWHAZ). Quantitative PCR (qPCR) experiments were performed on CFX Touch real-time PCR (Bio-Rad) in triplicate, including the positive and non-template controls. All procedures were carried out under stringent conditions to avoid contamination. A relative expression analysis was performed by qBase+ software [Version: 3.0, Biogazelle], using a comparative cycle threshold method (20 (link)). The change in gene expression fold compared with baseline (delta fold) was calculated for each subject.

At enrolment and after the rehabilitation program, whole blood (10 mL) was collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson & Co., Rutherford, NJ, USA). Peripheral blood mononuclear cells (PBMCs) were separated using the lympholyte separation medium (Cedarlane, Hornby, Ontario, CA, USA) and washed twice in PBS at 1500 rpm for 10 min; viable leukocytes were determined using a Scepter 2.0 Handheld Automated Cell Counter (Millipore, Billerica, MA, USA).