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: The following fluorophores, cloned into the vector [LeGO-G2], were used: EBFP2 (blue; abbreviated as “B”; plasmid 14891; Addgene) , T-Sapphire (violet excitable green; abbreviated as “S”) , and mOrange2 (orange; abbreviated as “O”; plasmid 30175; Addgene) ( ).

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

Journal: bioRxiv

Article Title: A Set of Novel Far-Red Fluorescent Proteins for Temporal Domain Multiplexing and Super-Resolution Imaging

doi: 10.1101/2025.06.01.657196

Figure Lengend Snippet: Characterization of mfRFP in mammalian cell culture. (a) Violin plot depicting far red-to-green fluorescence ratio in live HEK cells co-expressing E2-crimson, mCardinal, mCarmine, mGarnet, mNeptune, and mfRFP with EGFP via P2A self-cleaving peptide, 2 independent transfections each; imaging conditions: RFPs excitation 594nm, emission 660nm; GFP: 594nm excitation, emission) boxplot: box range; 25th and 75th percentile, center white dot; median, width density; frequency, black dots; outliers. (b) Normalized photobleaching curve for E2-crimson, mCardinal, mCarmine, mGarnet, mNeptune, and mfRFP expressed in HEK cells, 2 independent transfection each; imaging conditions for HEK cells: 594nm excitation, 660nm emission, power 20%, exposure time 20ms) (c) A Violin plot depicting far red-to-green fluorescence ratio in fixed HeLa cells co-expressing E2-crimson, EmiRFP870, mCardinal, mCarmine, mNeptune, and mfRFP with EGFP via P2A self cleaving peptide, 2 independent transfections each; imaging conditions: RFPs excitation 594nm, emission 660nm; GFP: 594nm excitation, emission) boxplot: box range; 25th and 75th percentile, center white dot; median, width density; frequency, black dots; outliers (d) Normalized photobleaching curve for E2-crimson, EmiRFP870, mCardinal, mCarmine, mNeptune, and mfRFP expressed in HEK cells; 2 independent transfection each; imaging conditions for HEK cells: 594nm excitation, 660nm emission, power 20%, exposure time 20 ms(c) (e) Representative images of mfRFP in various fusions (clockwise from top left) EB3-mfRFP, Mito-mfRFP, Keratine-mfRFP and H2B-mfRFP (n>5 cells for each construct from two independent transfections). Scale bar 25μm EB3,CytERM,Mito and H2B were obtained under identical microscopic setup Nikon Confocal 100X 500us exposure time at 594nm laser channel while Keratine was obtained with Nikon Confocal 500us exposure time at 594nmSoRa laser channel and Mito was obtained using the Olympus Confocal microscope 60X. (f) Confocal (top) and STED (bottom) images of H2B-mfRFP fusion. Scale bar 25 um. (g) Cross sections of the nucleus marked in orange line in (f), the line profile of the confocal (black, dot) and STED (dash, gray) image obtained from the solid orange line, the solid lines (red, confocal) (green, STED) represent Gaussian fits used to determine the full width at half-maximum (FWHM) values. (h) Confocal (top) and STED (bottom) images of Mito-mfRFP fusion. Scale bar 25 um. (i) Cross sections of the nucleus marked in orange line in (f), the line profile of the confocal (black, dot) and STED (dash, gray) image obtained from the solid orange line, the solid lines (red, confocal) (green, STED) represent Gaussian fits used to determine the full width at half-maximum (FWHM) values

Article Snippet: First, EBFP2 in pAAV-CAG-EBFP2-P2A-GFP (Addgene plasmids #184938) was swapped with the gene of the selected.

Techniques: Cell Culture, Fluorescence, Expressing, Transfection, Imaging, Construct, Microscopy