Ficoll paque premium density gradient media

All experiments and methods were performed in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Fukushima Medical University Institutional Review Board. All experimental procedures involving human samples were approved by the following ethics committees: the Human Genome and Gene Analysis Research Committee at Fukushima Medical University (approval number 2186), the Epidemiological and General Research Committee of the Faculty of Life Science, Kumamoto University, the Human Genome and Gene Analysis Research Committee of the Faculty of Life Sciences, Kumamoto University, and the Clinical Research and Advanced Medical Technology Committee, Kumamoto University (approval numbers 318, 153 and 1,018, respectively). Blood samples (10 ml) were collected from the two patients involved in this study, and T-cells were isolated using Ficoll-Paque PREMIUM density gradient media (GE Healthcare). Informed consent was obtained from the participant or the participant’s parents prior to the study.

SSC populations were selected by magnetic separation (STRO-1 or CD271) from adherent mononuclear cell fractions from human bone marrow obtained during routine knee/hip replacement surgeries. Briefly, bone marrow aspirate was thinned with basal media (DMEM supplemented with 10% (v/v) FBS, 1% (v/v) sodium pyruvate, 1% (v/v) non-essential amino acids, 1% (v/v) penicillin-streptomycin, 1% (v/v) l-glutamine, 5% (v/v) Fungizone^® amphotericin B), and layered onto Ficoll Paque Premium density gradient media (GE Healthcare). Following centrifugation at 1513g for 45 min, the intermediate interface of mononuclear cells was removed and washed with media successively for a total of three washes. The resulting pellet was transferred into a tissue culture flask and cells were cultured to near confluence. These were then detached and selected for the marker CD271 using a human CD271 positive selection kit and magnet (EasySep, Stem Cell Technologies) as per the manufacturer's instructions. STRO-1⁺ SSCs were selected similarly using an in-house STRO-1 antibody (hybridoma courtesy of Dr Beresford, University of Bath) using a MACS kit from Miltenyi Biotech. Over the course of this study, cells from 12 donors were used with each biological test using new cells.

PBMC were isolated from 5-ml of blood collected from the ear central artery before and at different time-points after infection. Immediately after euthanasia, single-cell suspensions were prepared from popliteal lymph node (pLN) and spleen as follows. Tissue biopsies were delicately chopped in sterile RPMI media and passed through a 70 μm cell-strainer (BD Biosciences). Mononuclear leukocyte suspensions from peripheral blood and tissue samples were prepared with Ficoll-Paque Premium density gradient media (GE Healthcare). 5-ml single-cell suspension was diluted 1:1 in sterile PBS, overlaid onto 5-ml Ficoll-Paque density cushion and centrifuged (1825×g) during 20-min at room temperature. Mononuclear leukocytes at the interface were collected and washed in ice-cold PBS before further analysis.