Journal: Stem Cell Reviews and Reports

Article Title: Inter- and Intrapopulational Heterogeneity of Characteristic Markers in Adult Human Neural Crest-derived Stem Cells

doi: 10.1007/s12015-021-10277-w

Figure Lengend Snippet: SMARTseq2 reveals strong transcriptional heterogeneity of single ITSCs in their NCSC-marker expression. A : ITSCs were derived from the inferior turbinate of the human nasal cavity. B : GFP-transfected ITCSs 24 h after transfection. C : Flow cytometric analyses of ITSCs. SSC and FSC measure size and granularity of the cells. Whole cells (44.1 %) are marked for further gating. D : Sorting of GFP + ITSCs. FL2 measures emitted fluorescence signal (GFP). Untransfected cells served as negative control. E : RT-PCR after SMARTseq2 for the housekeeping genes GAPDH, eEF2 and Vimentin as well as for the NCSC-markers S100, Snail and Twist. F - K : RT-PCRs after application of SMARTseq2 on manually diluted single cells from three male and three female donors for the housekeeping gene GAPDH and the stem cell and neural crest markers Nestin, Slug, Snail and Twist

Article Snippet: Primary antibodies against Nestin (Mouse, 10C2, 1:300, Merk KGaA, Germany), S100 (Rabbit, Z0311, 1:400, Dako Cytomation, Germany), Slug (Rabbit, C19G7, 1:100, Cell Signaling, Germany) or NF-κB-p65 (Mouse, 200,301,065, 1:5000, Rockland, PA USA) were incubated for 1 h at RT.

Techniques: Marker, Expressing, Derivative Assay, Transfection, Fluorescence, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Stem Cell Reviews and Reports

Article Title: Inter- and Intrapopulational Heterogeneity of Characteristic Markers in Adult Human Neural Crest-derived Stem Cells

doi: 10.1007/s12015-021-10277-w

Figure Lengend Snippet: Immunocytochemistry confirmed heterogeneous expression of Nestin and S100 between ITSCs in dependence to the donor. A : Schematic view on interpopulational heterogeneity in stem cell marker expression between individual ITSC-populations in dependence to the donor. B - C : Representative images of immunocytochemical stained Nestin protein in female and male-derived ITSCs. D : Quantification of immunocytochemistry via measurement of the cytosolic fluorescence intensity of Nestin per single cell validated a significant heterogeneity dependent on the donor on protein level. E - F : Representative images of immunocytochemical stained S100 protein in female and male-derived ITSCs. G : Quantification of immunocytochemistry via measurement of the cytosolic fluorescence intensity of S100 per single cell affirmed the heterogeneity of S100 expression on protein level. Kruskal-Wallis test, Post test: Dunn’s Multiple Comparison Test, *p<0.05, *p<0.01, *** p < 0.001. was considered significant

Article Snippet: Primary antibodies against Nestin (Mouse, 10C2, 1:300, Merk KGaA, Germany), S100 (Rabbit, Z0311, 1:400, Dako Cytomation, Germany), Slug (Rabbit, C19G7, 1:100, Cell Signaling, Germany) or NF-κB-p65 (Mouse, 200,301,065, 1:5000, Rockland, PA USA) were incubated for 1 h at RT.

Techniques: Immunocytochemistry, Expressing, Marker, Staining, Derivative Assay, Fluorescence, Comparison

Journal: Stem Cell Reviews and Reports

Article Title: Inter- and Intrapopulational Heterogeneity of Characteristic Markers in Adult Human Neural Crest-derived Stem Cells

doi: 10.1007/s12015-021-10277-w

Figure Lengend Snippet: Stochastic single cell heterogeneity within clonally grown ITSC-culture. A : Schematic view on heterogeneous protein amounts between single ITSCs in the same clonal culture. B : RT-PCR revealing transcriptional heterogeneity of clonally grown ITSCs regarding the expression of NCSC-markers Nestin, Slug, Snail and Twist. C , E , G , I : Representative images of immunocytochemical stainings showing heterogeneity of Nestin, S100, Slug and NF-κB-p65 proteins between single ITSCs present in the same clonally grown culture. D , F , H , J : Quantification of immunocytochemical stainings via measurement of the single cell or single nuclear fluorescence intensity showing the stochastic variability in Nestin, S100, Slug and NF-κB-p65 protein amounts in clonally grown ITSCs

Article Snippet: Primary antibodies against Nestin (Mouse, 10C2, 1:300, Merk KGaA, Germany), S100 (Rabbit, Z0311, 1:400, Dako Cytomation, Germany), Slug (Rabbit, C19G7, 1:100, Cell Signaling, Germany) or NF-κB-p65 (Mouse, 200,301,065, 1:5000, Rockland, PA USA) were incubated for 1 h at RT.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Fluorescence

Journal: Frontiers in Immunology

Article Title: Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity

doi: 10.3389/fimmu.2023.1225610

Figure Lengend Snippet: Purification of novel anti-CD30 mAbs and binding to huCD30. (A) Coomassie Blue-stained gel following non-reducing PAGE of purified 8D10 and 8D10F10 (subclone for preparation of a clinical trial MCB). (B) Binding of various dilutions of our novel mAbs to huCD30-GST protein was assessed by ELISA. (C) FCM analyses were carried out for all five novel CD30 mAbs and AC10. Representative FCM histograms are shown.

Article Snippet: Mouse CD30 hybridomas (clones 8D10, 10C2, 12B1, 13H1, 15B8) (all custom generated by Genscript), were derived by fusing murine Sp2/0 myeloma cells with spleen cells from a BALB/c mouse that had been immunized with purified huCD30-GST and boosted.

Techniques: Purification, Binding Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity

doi: 10.3389/fimmu.2023.1225610

Figure Lengend Snippet: Affinity constants for the interaction of CD30 mAbs with immobilized huCD30 protein.

Article Snippet: Mouse CD30 hybridomas (clones 8D10, 10C2, 12B1, 13H1, 15B8) (all custom generated by Genscript), were derived by fusing murine Sp2/0 myeloma cells with spleen cells from a BALB/c mouse that had been immunized with purified huCD30-GST and boosted.

Techniques:

Journal: Frontiers in Immunology

Article Title: Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity

doi: 10.3389/fimmu.2023.1225610

Figure Lengend Snippet: Categorization of mAbs by sequence and cluster binding. (A) V L and V H protein sequences were compared across all 5 novel mAbs and AC10. Levels of sequence similarity range from white (low) to blue (high). Numbering indicates the sequence identity as a percentage. (B) Cluster mapping via competitive binding. Fluorescently labelled mAbs were incubated with CD30 + SU-DHL-1 cells blocked with excess unlabeled mAbs. FCM analysis was then performed. High inhibition (red) indicates a shared cluster and low inhibition (blue) indicates distinct clusters. Results represent the means of 3 independent experiments (n=3). (C) A depiction of the three known clusters of mAb binding to CD30.

Article Snippet: Mouse CD30 hybridomas (clones 8D10, 10C2, 12B1, 13H1, 15B8) (all custom generated by Genscript), were derived by fusing murine Sp2/0 myeloma cells with spleen cells from a BALB/c mouse that had been immunized with purified huCD30-GST and boosted.

Techniques: Sequencing, Binding Assay, Incubation, Inhibition

Journal: Frontiers in Immunology

Article Title: Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity

doi: 10.3389/fimmu.2023.1225610

Figure Lengend Snippet: Assessment of biAb binding capabilities. (A) BiAbs capable of binding to CD3 and CD30 were developed by heteroconjugation of mAb 8D10 or 10C2 with OKT3. (B) The ability of biAbs 8D10 and 10C2 to bind to both CD3 and CD30 post-conjugation was determined by FCM. Binding of unconjugated 8D10, 10C2, and OKT3 mAbs and an isotype control is shown for reference. (C) A two-color FCM assay was developed to determine T cell-Raji LV30 conjugate formation in the presence of biAbs. (D) Assessment of percent conjugation of Raji and Raji LV30 cells with unarmed or biAb-armed T cells was measured by detection of two-color events and plotted as a percentage of all events. Results represent means ± SD of 3 independent experiments (n = 3). Representative FCM plots from this assay are shown in (E) . n.s, not significant, ** = p < 0.01 between the indicated groups, calculated using a one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Mouse CD30 hybridomas (clones 8D10, 10C2, 12B1, 13H1, 15B8) (all custom generated by Genscript), were derived by fusing murine Sp2/0 myeloma cells with spleen cells from a BALB/c mouse that had been immunized with purified huCD30-GST and boosted.

Techniques: Binding Assay, Conjugation Assay, Control

Journal: Frontiers in Immunology

Article Title: Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity

doi: 10.3389/fimmu.2023.1225610

Figure Lengend Snippet: Cytokine production by biAb-armed T cells after co-culture with tumor cells. (A) Co-culture conditions included no target (0), a CD30 low cell line (Raji), CD30 + cell lines (Raji LV30 and RPMI 6666), and a CD30 − cell line (OCIAML2). Production of IFN-γ, TNF-α, IL-2, GM-CSF, and IL-4 was assessed. (B) Co-culture conditions included no target (0), a CD30 low cell line (Raji), and a CD30 + cell line (HH). Results represent means ± SD of 3 independent experiments (n = 3). * = p < 0.05, ** = p < 0.01, *** = p < 0.001 between the indicated groups, calculated using a two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Mouse CD30 hybridomas (clones 8D10, 10C2, 12B1, 13H1, 15B8) (all custom generated by Genscript), were derived by fusing murine Sp2/0 myeloma cells with spleen cells from a BALB/c mouse that had been immunized with purified huCD30-GST and boosted.

Techniques: Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity

doi: 10.3389/fimmu.2023.1225610

Figure Lengend Snippet: Cytotoxicity of CD30/CD3 biAb-armed T cells. (A–F) Cytotoxicity of 8D10 & 10C2 biAb-armed T cells against CD30 − and CD30 + tumor cells was assessed using a standard 4-hour 51 Cr release assay. Lysis of two tumor cell lines with endogenous CD30 expression, (A) SU-DHL-1 and (B) RPMI 6666, was measured, in addition to (C) OCIAML2, a CD30 − cell line, (D) NT Raji, a CD30 low cell line, and (E) Raji LV30 cells, which are CD30 high . (F) Activated T cells, which express low levels of CD30, were also assessed for lysis. Cytotoxicity of unarmed, 8D10, and AC10 biAb-armed T cells was compared at 24 hours against Raji LV30 cells (G) after T cells were armed with varying levels (50-500ng/million cells) of biAb. Cytotoxicity of 8D10 and 8D10F10 biAb-armed human T cells used fresh or after cryopreservation against CD30 + cell lines: (H) HH, (I) Karpas, and (J) L428 was ascertained. Results represent means ± SD of 3-4 independent experiments per cell line (n = 3-4). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 between biAb-armed T cells and unarmed T cells. # = p < 0.05, ## = p < 0.01, #### = p < 0.0001 between 8D10 and 10C2 biAb-armed T cells (A–F) and between 8D10 and AC10 biAb-armed T cells (G) . Statistical significance was calculated using a two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Mouse CD30 hybridomas (clones 8D10, 10C2, 12B1, 13H1, 15B8) (all custom generated by Genscript), were derived by fusing murine Sp2/0 myeloma cells with spleen cells from a BALB/c mouse that had been immunized with purified huCD30-GST and boosted.

Techniques: Release Assay, Lysis, Expressing

Journal: Acta Endocrinologica (Bucharest)

Article Title: THE EXPRESSION OF STEM CELL MARKERS (CD133, NESTIN, OCT4, SOX2) IN INVASIVE PITUITARY ADENOMAS

doi: 10.4183/aeb.2020.303

Figure Lengend Snippet: Patient and tumor characteristics, including histopathological staining results

Article Snippet: Surgical specimens were fixed with 4% paraformaldehyde; paraffin-embedded sections were mounted on glass slides, deparaffinized, and reacted with either a CD133 rabbit polyclonal antibody (Catalog no: {"type":"entrez-protein","attrs":{"text":"ORB18124","term_id":"1177139486","term_text":"ORB18124"}} ORB18124 ; Biorobot Limited, Cambridge,UK), a nestin rat monoclonal antibody (Clone no: 10C2; Gene TexInc, Irvine, CA,US), an OCT4 rabbit polyclonal antibody (Subclass: IgG; GeneTexInc, Irvine, CA,US), or a SOX2 rabbit polyclonal antibody (Clone No: NT2D1; Gene TexInc; Irvine,CA,US) as the primary antibody (dilution 1:100).

Techniques: Staining

Journal: Acta Endocrinologica (Bucharest)

Article Title: THE EXPRESSION OF STEM CELL MARKERS (CD133, NESTIN, OCT4, SOX2) IN INVASIVE PITUITARY ADENOMAS

doi: 10.4183/aeb.2020.303

Figure Lengend Snippet: a) Diffuse cytoplasmic expression of CD133 (original magnification x400, b) Diffuse cytoplasmic expression of nestin (original magnification x400), c) Diffuse nuclear expression of OCT-4 (original magnification x400), and d) Patchy nuclear expression of SOX-2 (original magnification x400) in pituitary adenoma cells.

Article Snippet: Surgical specimens were fixed with 4% paraformaldehyde; paraffin-embedded sections were mounted on glass slides, deparaffinized, and reacted with either a CD133 rabbit polyclonal antibody (Catalog no: {"type":"entrez-protein","attrs":{"text":"ORB18124","term_id":"1177139486","term_text":"ORB18124"}} ORB18124 ; Biorobot Limited, Cambridge,UK), a nestin rat monoclonal antibody (Clone no: 10C2; Gene TexInc, Irvine, CA,US), an OCT4 rabbit polyclonal antibody (Subclass: IgG; GeneTexInc, Irvine, CA,US), or a SOX2 rabbit polyclonal antibody (Clone No: NT2D1; Gene TexInc; Irvine,CA,US) as the primary antibody (dilution 1:100).

Techniques: Expressing

Journal: Acta Endocrinologica (Bucharest)

Article Title: THE EXPRESSION OF STEM CELL MARKERS (CD133, NESTIN, OCT4, SOX2) IN INVASIVE PITUITARY ADENOMAS

doi: 10.4183/aeb.2020.303

Figure Lengend Snippet: Distribution of pituitary stem cells staining features according to histology of the adenomas

Article Snippet: Surgical specimens were fixed with 4% paraformaldehyde; paraffin-embedded sections were mounted on glass slides, deparaffinized, and reacted with either a CD133 rabbit polyclonal antibody (Catalog no: {"type":"entrez-protein","attrs":{"text":"ORB18124","term_id":"1177139486","term_text":"ORB18124"}} ORB18124 ; Biorobot Limited, Cambridge,UK), a nestin rat monoclonal antibody (Clone no: 10C2; Gene TexInc, Irvine, CA,US), an OCT4 rabbit polyclonal antibody (Subclass: IgG; GeneTexInc, Irvine, CA,US), or a SOX2 rabbit polyclonal antibody (Clone No: NT2D1; Gene TexInc; Irvine,CA,US) as the primary antibody (dilution 1:100).

Techniques: Staining, Cell Counting