Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Phenotyping clonal populations of glioma stem cell reveals a high degree of plasticity in response to changes of micro-environment

doi: 10.1038/s41374-021-00695-2

Figure Lengend Snippet: A , combinations of 4 lentiviruses expressing EBFP2 (B [blue]), T-Sapphire (S), Venus (V) and mOrange2 (O) results in 6 populations of double labelled cells, BS, BV, BO, SV, SO, or VO. B , spectra of the fluorescent proteins and the surface markers, showing the fluorophores at the left of the spectrum, separated from the surface markers for expression phenotyping on the right of the spectrum. Spectral diagram from . C , Flow cytometry detection of 6 different labels in a mixed (n ~ 76,000) population derived from a cell culture one passage after FACS purification, eliminating single labelled cells. Axis dimensions set to demonstrate wide spread of label detections in each colour group. The right panel shows examples of fluorescent images of double labelled cells. D , quantification of colour group frequencies from flow cytometry data (n = 3) for cell lines G19 and G61. E , experimental workflow encompassing all experimental steps including organoid formation. Experimental steps (“ES”) are indicated as ES 1-ES 6, to provide a generic reference in the text. Naïve cells (G19 and G61) were first analysed for surface marker expression (ES 1), and then transduced with lentivirus encoding two fluorescent labels and incubated to express them (ES 2), analysed by flow cytometry for clonality and surface marker expression (ES 3), propagated in 6 parallel cultures, over 5 passages (P1-P5) (ES 4, ES 5, ES 6), with 3 flow cytometry and surface marker measurements performed at ES 4, 5, and 6. Finally, two of the clonal cultures were processed to generate organoids (ES 7). In addition, mixed organoids were generated directly from mixed dual labelled cells containing all label combinations before clonal segregation (left part of diagram). Scale bar in A corresponds to 10 μm.

Article Snippet: G61 and G19 dual labelled cells (BS, BV, BO, SV, SO, VO) were expanded in adherent culture, detached and dissociated to single cell suspension (Accutase ® , Sigma-Aldrich A6964) in flow cytometry pre-sort buffer (BD Biosciences, 5636503).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Cell Culture, Purification, Marker, Transduction, Incubation, Generated

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Phenotyping clonal populations of glioma stem cell reveals a high degree of plasticity in response to changes of micro-environment

doi: 10.1038/s41374-021-00695-2

Figure Lengend Snippet: A , flow cytometry of barcode labelled cells (G61) before seeding at P0, (ES 2). Expansions of cells into subcultures C1-C6 and formation of clonal population visible as “clouds” or “streaks” during propagation. Clonal formation in culture C3 (ES 2-ES 6), corresponding to passages P2, P3, and P5. B , confocal imaging of cultures C1-C6 at P2 and P5, showing formation of emerging clonal populations of barcoded cells (ES 4, 6). Scale bar corresponds to 50 μm.

Article Snippet: G61 and G19 dual labelled cells (BS, BV, BO, SV, SO, VO) were expanded in adherent culture, detached and dissociated to single cell suspension (Accutase ® , Sigma-Aldrich A6964) in flow cytometry pre-sort buffer (BD Biosciences, 5636503).

Techniques: Flow Cytometry, Imaging

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Phenotyping clonal populations of glioma stem cell reveals a high degree of plasticity in response to changes of micro-environment

doi: 10.1038/s41374-021-00695-2

Figure Lengend Snippet: A , P5 culture with a predominance of clonal populations with barcode label BS and BO (cell line G61) to generate organoids. B , Flow cytometry of dissociated organoids show the two dominant label populations BS and BO. Arrowheads indicate clonal populations which continue their dominance in organoids. C , D , E , 3 separate organoids grown from cells with colour barcodes BS and BO, 14 days after seeding, showing both populations of barcoded cells populating the organoid in specially organised patterns. Arrowheads in C - E point to populations of barcoded clones. F - K , 3 separate organoids (surface aspect) ( F , G , H ), and the same organoids centrally cross-sectioned ( I , J , K ) showing spatially organised BS and BO clones as indicated by arrowheads in F and G . L , These label populations were then analysed for surface marker expression showing A2B5-dominant populations overall, whilst separate analysis of label barcoded clones shows surface marker separation when analysing clones BS and PO separately, with a marked difference of A2B5 expression, and a notable dominance of cells negative for all surface marker in the BS clone. Colour codes see and . M , proportion of barcoded clonal populations across 10 separately analysed organoids. Scale bar in C - K corresponds to 500 μm.

Article Snippet: G61 and G19 dual labelled cells (BS, BV, BO, SV, SO, VO) were expanded in adherent culture, detached and dissociated to single cell suspension (Accutase ® , Sigma-Aldrich A6964) in flow cytometry pre-sort buffer (BD Biosciences, 5636503).

Techniques: Flow Cytometry, Clone Assay, Marker, Expressing