fluorescent mounting media with dapi  (OriGene)

Journal: Cell reports

Article Title: G2 arrest primes hematopoietic stem cells for megakaryopoiesis

doi: 10.1016/j.celrep.2024.114388

Figure Lengend Snippet: (A) Flow cytometric analysis of BM from control mice and from mice 24 h after 450-cGy X-ray irradiation ( n = 3). (B) Quantification of (A) gated on HSCs ( n = 3, each experiment averaged across technical replicates, unpaired t tests [Mann-Whitney for CD9]). (C) Confocal images of GATA1-stained HSCs isolated from control mice or 24 h after 450-cGy X-ray irradiation ( n = 3). (D) Quantification of percentage of GATA1 + cells and GATA1 integrated density in HSCs isolated from control mice or 24 h after 450-cGy X-ray irradiation ( n = 3, unpaired t test for fraction of GATA1, and Mann-Whitney test for GATA1 IntDens). (E) Confocal images of HSCs isolated 24 h after PBS or poly(I:C) treatment and stained for RPA32, γH2A.X, and DAPI. (F) Quantification of RPA32 and γH2A.X integrated density, and percentage of RPA32 + cells ( n = 3 independent sorts; each point an individual cell for RPA32 and γH2A.X integrated density; each point 1 individual experiment for percentage of RPA32 + cells). Error bars represent mean ± SEM. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001.

Article Snippet: After three additional washes in PBS, stained cells were mounted in fluorescent mounting media with DAPI (OriGene, Rockville, MD) and stored at 4° C in tinfoil until ready to image.

Techniques: Control, Irradiation, MANN-WHITNEY, Staining, Isolation

Journal: Cell reports

Article Title: G2 arrest primes hematopoietic stem cells for megakaryopoiesis

doi: 10.1016/j.celrep.2024.114388

Figure Lengend Snippet: (A) Bright-field images of vehicle- and idarubicin-treated 48-h HSC cultures (representative of 5 experiments). (B) Flow cytometric analysis of MK surface markers in idarubicin- or DMSO vehicle-treated 48-h HSC cultures ( n = 5, each averaged across 3 technical replicates and normalized to vehicle, 1-sample t test). (C) Representative confocal images of HSCs stained for CD41 and DAPI after 48-h culture with vehicle or idarubicin culture (representative of 3 experiments). (D) Fraction of CD41 + cells as evaluated by confocal microscopy ( n = 3, paired t test). (E) Confocal images of HSCs stained for GATA1 and DAPI after 48-h culture with vehicle or idarubicin culture (representative of 3 experiments). (F) Quantification of percentage of GATA1 cells after 48-h culture of HSCs with vehicle or idarubicin ( n = 3, unpaired t test). (G) Quantification of GATA1 integrated density within GATA1 + population ( n = 3, unpaired t test). (H) qRT-PCR for MK markers in 48-h vehicle- and idarubicin-treated HSC cultures (normalized to vehicle, n = 5 unpaired t test). (I) Flow cytometry histograms of cell cycle/ploidy in 48-h HSC cultures with vehicle or idarubicin (representative of 5 experiments). (J) Quantification of cell cycle/ploidy analyses in 48-h HSC cultures with vehicle or idarubicin ( n = 5, paired t test). (K) Flow cytometry histograms of propidium iodide cell-cycle and ploidy analyses in 48-h HSC cultures with vehicle, idarubicin, and/or adavosertib (representative of 3 experiments). (L) Quantification of fraction sub-G1 and G2 cells in 48-h HSC cultures with vehicle, idarubicin, and/or adavosertib ( n = 3, 1-way ANOVA with Tukey’s multiple comparisons test). (M) Flow cytometric analysis of MK surface markers in 48-h HSC cultures with vehicle, idarubicin, and/or adavosertib ( n = 3, each experiment averaged across technical replicates, 1-way ANOVA with Holm-Sidak’s multiple comparisons). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p < 0.0001. Error bars represent mean ± SEM.

Article Snippet: After three additional washes in PBS, stained cells were mounted in fluorescent mounting media with DAPI (OriGene, Rockville, MD) and stored at 4° C in tinfoil until ready to image.

Techniques: Staining, Confocal Microscopy, Quantitative RT-PCR, Flow Cytometry

Journal: Cell reports

Article Title: G2 arrest primes hematopoietic stem cells for megakaryopoiesis

doi: 10.1016/j.celrep.2024.114388

Figure Lengend Snippet: (A) Expression of MK surface markers in RO-3306- or vehicle-treated 48-h cultures of CD41 + and CD41 − HSCs ( n = 3, each experiment averaged across technical replicates, unpaired t test). (B) Fraction GATA1 + cells in CD41 − and CD41 + HSCs ( n = 3, unpaired t test). (C) γH2A.X integrated density in fresh CD41 − and CD41 + HSCs ( n = 3, Mann-Whitney test). (D) Confocal images of fresh CD41 − and CD41 + HSCs stained for γH2A.X and GATA1 (representative of 3 experiments). (E) Quantification of γH2A.X in GATA1 + and GATA1 − CD41 − and CD41 + HSCs ( n = 3, unpaired t test for CD41 − HSCs, and Mann-Whitney tests for CD41 + HSCs). (F) Confocal images of 48-h HSC cultures in RO-3306 and vehicle control, stained for γH2A.X (green) ( n = 1). (G) Quantification of nuclear γH2A.X integrated density normalized to cell area (pixel2) based on DAPI stain ( n = 1, Mann-Whitney test). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. Error bars represent mean ± SEM.

Article Snippet: After three additional washes in PBS, stained cells were mounted in fluorescent mounting media with DAPI (OriGene, Rockville, MD) and stored at 4° C in tinfoil until ready to image.

Techniques: Expressing, MANN-WHITNEY, Staining, Control

Journal: Cell reports

Article Title: G2 arrest primes hematopoietic stem cells for megakaryopoiesis

doi: 10.1016/j.celrep.2024.114388

Figure Lengend Snippet: (A) Semi-quantitative determination of uracil content in DNA (Frac U DNA ) in fresh and 7-day cultured Ung −/− HSCs and macrophages (MAC), and in HSCs culture in the presence of thymidine (100 μM) ( n = 3, 1-way ANOVA). (B) Frac U DNA in HSCs and lin + cells 24 h after administration of poly(I:C) in vivo ( n = 6, unpaired t test). (C) Confocal images of resorted HSCs from 7-day HSC cultures with or without thymidine stained for γH2A.X and DAPI (representative of 3 experiments). (D) Quantification of nuclear γH2A.X in fresh and 7-day resorted HSCs from culture with and without thymidine ( n = 3, 1-way ANOVA). (E) Confocal images of resorted HSCs from 7-day HSC cultures with or without thymidine stained for RPA32 and DAPI (representative of 3 experiments). (F) Quantification of RPA32 expression in fresh and 7-day resorted HSCs from HSC cultures with and without thymidine ( n = 3, unpaired t test). (G) CD41 geometric mean fluorescence intensity (MFI) in fresh and 7-day cultured HSCs ( n = 3, unpaired t test). (H) CD41 geometric MFI of HSCs and LSKs after 7-day culture with or without thymidine ( n = 3, unpaired t tests). (I) 16-week primary and secondary post-transplantation chimerism of fresh HSCs and HSCs cultured with or without thymidine for 14 days ( n = 3, 1-way ANOVA). (J) KuO + total, platelet, and red blood cell PB chimerism after competitive transplantation of 14-day HSC cultures with vehicle orthymidine ( n = 5, unpaired t test). Error bars represent mean ± SEM.

Article Snippet: After three additional washes in PBS, stained cells were mounted in fluorescent mounting media with DAPI (OriGene, Rockville, MD) and stored at 4° C in tinfoil until ready to image.

Techniques: Cell Culture, In Vivo, Staining, Expressing, Fluorescence, Transplantation Assay

Journal: Cell reports

Article Title: G2 arrest primes hematopoietic stem cells for megakaryopoiesis

doi: 10.1016/j.celrep.2024.114388

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After three additional washes in PBS, stained cells were mounted in fluorescent mounting media with DAPI (OriGene, Rockville, MD) and stored at 4° C in tinfoil until ready to image.

Techniques: Recombinant, Adhesive, Purification, Blocking Assay, Amplification, Random Hexamer, Reverse Transcription, SYBR Green Assay, Sterility, Imaging, Software