Collagenase d

Single-cell suspensions from mouse spleen tissue were obtained by digestion with (FACS) or without (endocytosis assay, ex vivo proliferation assay) Collagenase D (Serva) and DNase I (Roche) as described before (Dudziak et al., 2007 (link)). Erythrocyte lysis was performed with ACK solution (Invitrogen), followed by filtering through a 40-µm mesh (BD) and washing in FACS (PBS, 2% FCS, and 0.09% NaN₃) or MACS buffer (PBS and 0.5% BSA, degased).

All of the chemicals, unless otherwise stated, were from Sigma-Aldrich (Taufkirchen, Germany). Citalopram was purchased from Biotrend (Cologne, Germany) and collagenase D was from Serva (Heidelberg, Germany). [³H]DHEAS (70.5 Ci/mmol), [³H]E-3-S (45.6 Ci/mmol), [³H]aspartate (11.3 Ci/mmol), [³H]GABA (76.2 Ci/mmol), [³H]histamine (13.4 Ci/mmol), [³H]choline chloride (66.7 Ci/mmol), [³H]norepinephrine (56.6 Ci/mmol), [³H]serotonin (28.25 Ci/mmol), [³H]dopamine (38.7 Ci/mmol), [³H]glutamate (49.6 Ci/mmol), [³H]ATP (30.9 Ci/mmol), [³⁵S]Adenosine 5′-(γ-thio) triphosphate (12.5 mCi/mmol) and [³H]acetylcholine iodide (99.7 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA, USA). [³H]PREGS (20 Ci/mmol), [³H]taurocholic acid (10.0 Ci/mmol), [³H]acetate (150 mCi/mmol), and [³H]lithocholic acid (50 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO, USA).

Leukocyte reduction cones were retrieved from anonymous healthy adult donors. Thymus samples were retrieved from cardiac surgeries of otherwise healthy children. The sources of spleen samples were patients requiring therapeutic splenectomy. All samples were received under local ethical committee approvals (Ethikkommission der Friedrich-Alexander-Universität Erlangen-Nürnberg), and informed written consents were obtained in accordance with the Declaration of Helsinki.
All tissues were freshly processed as described earlier (15 (link)). In brief, thymic and splenic tissues were chopped into small pieces using forceps and scalpel. Then, the tissue was transferred into C-tubes (Miltenyi Biotec), filled with 5 ml RPMI1640, further mechanically disrupted using a Gentle MACS tissue dissociator (Miltenyi Biotec), and enzymatically digested with 400 U/ml collagenase D (Serva) and 100 µg (spleen) or 300 µg (thymus) deoxyribonuclease I (Sigma). After filtering the cell suspension twice, cell suspension of splenic and thymic tissue as well as the leukocyte enriched fraction of human blood was diluted with RPMI1640 and a density gradient centrifugation using Human Pancoll (ρ = 1.077 g/ml; Pan Biotech) was performed as described earlier. After the centrifugation, the interphase containing the mononuclear cells was collected, washed twice with RPMI1640, and used for experiments.