Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-gradient centrifugation (Biochrom, Berlin, Germany). Total Treg and Tcon were highly enriched from freshly isolated PBMCs by means of immunomagnetic separation utilizing Dynabead technology as previously described (19 (link), 27 (link), 28 (link)).
Ficoll gradient centrifugation
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Ficoll-gradient centrifugation is a laboratory technique used to separate and isolate different types of cells or cellular components based on their density. It involves the use of a Ficoll solution, a synthetic polymer, which creates a density gradient during centrifugation. This allows the separation of various cell populations or organelles, such as lymphocytes, monocytes, or mitochondria, based on their specific densities.
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5 protocols using ficoll gradient centrifugation
1
Isolation and Enrichment of Regulatory T Cells
2
Peripheral Blood and CSF Sampling
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From all study participants, 10–50 mL peripheral blood and 10 mL serum were collected. Sera were immediately stored at −70°C. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by Ficoll-gradient centrifugation (Biochrom, Berlin, Germany). Parallel CSF samples from 12 patients with pedMS and from 20 patients with adMS (0.5–3.0 mL) were obtained by lumbar puncture, immediately placed on ice, and sedimented for 10 minutes at 300 g and 4°C. Freshly isolated CSF cells were analyzed by flow cytometry. Supernatants were snap-frozen and stored at −70°C.
3
Isolation and Stimulation of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll gradient centrifugation (Biochrom AG, Berlin, Germany) and resuspended in RPMI medium supplemented with 2 mM l -glutamine, 1% non-essential amino acids, 100 U/mL penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated pooled human AB serum (CC Pro, Oberdorla, Germany) and stimulated at 6 h of culture with 100 ng/ml LPS (Escherichia coli 026:B6; Sigma-Aldrich) supplemented with 1 µg/ml brefeldin A (Sigma-Aldrich) to inhibit cytokine secretion and cultured for a total of 24 h (37°C at 5% CO2). For detection of TNF-α in slanMo before and after participation in the stress test, blood was taken, PBMCs were stimulated with LPS for 6 h, and subsequently stained for TNF-α.
4
Isolation of Bone Marrow Mononuclear Cells
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Fresh peripheral blood samples were obtained preoperatively and processed as described previously.8 Following general anesthesia, the right or left anterior superior iliac crest of the patient, who was lying in a supine position, was punctured with a bone marrow biopsy needle (Illinois needle, DIN1518X, CareFusion, Waukegan, IL) under sterile conditions. Thirty-five milliliters of bone marrow blood was aspirated and anticoagulated with 5 mL of heparin (Heparin-Natrium 25000 I.E./5 ml, Leo Pharma, Neu-Isenburg, Germany). The samples were immediately subjected to Ficoll gradient centrifugation (Biochrom, Berlin, Germany), and the cells in the interface were collected as previously described.8 ,42
5
Isolation and Purification of B Cells from PBMC and CSF
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From all study participants peripheral blood mononuclear cells (PBMCs) were isolated from 10 to 50 ml peripheral blood by Ficoll-gradient centrifugation (Biochrom, Berlin, Germany). Cerebrospinal fluid (CSF) samples (0.5–4.5 ml) were obtained from a subcohort of eight study patients (all in acute relapse and treatment-naïve). Total B cells were purified from PBMCs and from CSF cells with CD19 Microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) or Dynabeads Untouched Human B cells Kit (Thermo Fisher, Dreieich, Germany).
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