Moflo sorter

Side population (SP) and non-SP cells were analysed and sorted from Huh7 cells as described before [14 (link)] by using Hoechst33342 staining and MoFlo sorter (DAKOCytomation). Expression of the enzyme aldehyde dehydrogenase (ALDH) was analysed by using the ALDEFLUOR Kit from Stemcell Technologies (Grenoble, France) according to manufacturer's protocol after 24 hrs exposure of Huh7 cells to MTBITC. Cell sorting was performed with a BD FACS Aria III Cell Sorter (San Jose, CA, USA).

Cells were trypsinized and dissociated to single‐cell suspension. Cells were pelleted at 1,000 g for 1 min, resuspended in D‐PBS, and strained through a 40‐μm filter. Cells were analyzed on an LSRFortessa flow cytometer (BD BioSciences). GFP‐positive and GFP‐negative cells as well as TagBFP‐positive and TagBFP‐negative cells were FACS‐purified using a MoFlo sorter (DakoCytomation) or an Aria Fusion sorter (BD BioSciences) during the differentiation of the Sox1‐Brachyury‐Eomes triple knock‐in mESC reporter line. Flow cytometry data were analyzed with FlowJo or custom Python scripts.

Explants from the nasal area (E14), both nasal and basal forebrain areas (E17) and from basal forebrain area (E20) were microdissected from heads of Gnrh1-GFP embryos; nose and forebrain cells were dissociated by incubation in 0.05% trypsin with 100 μg/ml DNase I in neurobasal medium (Invitrogen) at 37°C for 15 min. Trypsinization was quenched by addition of neurobasal medium containing 10% heat-inactivated FBS (Invitrogen) at 37°C for 5 min. Cells were washed three times in neurobasal medium (without FBS) to remove serum before FACS and resuspended in neurobasal medium without Phenol Red (Invitrogen) containing L-glutamine (Invitrogen) and B-27 supplement (1:50; Invitrogen). Dissociated cells from eight to ten embryos for each age were pooled for each FACS. FACS was performed by the Wolfson Scientific Support Services (University College London, London, UK) by using a MoFlo Sorter (Dako). A non-green embryo was used as a control for fluorescence. Cells were excited by using a 488-nm Argon laser and detected by using a 530/40 (FL1) bandpass filter. A cell purity of 95-98.5% was obtained for each sort. Sorted (GFP⁺) and unsorted (GFP⁻) cells were directly collected in lysis buffer (Qiagen) and used for RNA extraction.

Assuming a Poisson distribution, the well with 5% GFP-infected cells was selected for expansion. This corresponds with a low MOI of ∼0.05 and as previously demonstrated ensures at most one integration event per infected cell within the population (Weinberger et al, 2005 (link)). The selected population was expanded for 7 days and stimulated with TNF-α as described above. Approximately 10⁵ cells were sorted from the GFP^; population on a DAKO-Cytomation MoFlo Sorter. The resulting population, which represents a polyclonal ensemble of single-integration clones, was expanded for 7 days. Single-cell clones were sorted from a wide gate distinct from background fluorescence into multi-well plates and cultured for 3–4 weeks to facilitate expansion.