ntb buffer  (MACHEREY NAGEL)

Journal: Blood Neoplasia

Article Title: Targeting the G-protein–coupled estrogen receptor: a novel therapeutic strategy in cutaneous T-cell lymphoma ∗

doi: 10.1016/j.bneo.2026.100213

Figure Lengend Snippet: G-1 induces apoptosis in CTCL cells. (A) For a 48-hour period, CTCL cells were exposed to either 500nM G-1 or DMSO. Cells were then stained for 20 minutes with Annexin V-FITC and 7-AAD. Annexin V–positive cells were analyzed by flow cytometry. Representative data shown from n = 3 independent experiments (left panel) and mean value of n = 3 independent experiments with SD (right panel). (B) The activation of caspase-3/7 by G-1 was assessed using the Caspase-Glo 3/7 assay. Cells were treated for 24 hours with 500nM G-1. The resulting caspase activity was normalized against cells treated with DMSO, which served as the untreated control. Mean values of n = 3 independent experiments with SD are plotted. (C) Different durations of treatment with DMSO or 500nM G-1 were applied to HuT and MyLa cells. Western blot analysis was used to evaluate protein expression levels. GAPDH served as a loading control. Representative plot of n = 3 independent experiments. Western blots were imaged digitally. Brightness and contrast were adjusted globally for each blot to improve clarity; no individual lanes were modified or removed. (D) Densitometric quantification of western blots shown in panel C. Band intensities were quantified using Image Studio, normalized to the loading control and expressed as fold change relative to DMSO (set to 1). Bars represent mean ± SD of n = 3 independent experiments. 7-AAD, 7-aminoactinomycin D; cIAP1, cellular inhibitor of apoptosis protein-1; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PARP, poly(ADP-ribose) polymerase; SD, standard deviation.

Article Snippet: After treatment, cells were collected, resuspended in Annexin V binding buffer, and incubated with Annexin V fluorescein isothiocyanate and 7-aminoactinomycin D (7-AAD; Miltenyi Biotech, Bergisch Gladbach, Germany) for 20 minutes at room temperature, protected from light.

Techniques: Staining, Flow Cytometry, Activation Assay, Caspase-Glo Assay, Activity Assay, Control, Western Blot, Expressing, Modification, Standard Deviation

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: CRISPR/Cas9 induces DNA damage and apoptosis in PGCs. (A) Flow cytometry analysis 24 h after electroporation, quantifying the proportion of Annexin V + /PI + cells. The horizontal axis indicates PI and the vertical axis Annexin V. The upper-left quadrant (Annexin V + /PI + ) represents late apoptotic cells, and the lower-right quadrant (Annexin V + only) represents early apoptotic cells. Upper panels: results after electroporation with Cas9 + various gRNAs; lower panels: results with dCas9 + various gRNAs. (B) Bar graph of Annexin V + /PI + percentages across groups. Cas9 editing induced a highly significant increase in late apoptosis. (C) γ-H 2 AX foci (green) detected by immunofluorescence 24 h after electroporation. Foci appear as discrete nuclear puncta; nuclei are counterstained with DAPI (blue). Scale bar = 10 µm. (D) Quantification of γ-H 2 AX foci per cell. Cas9 targeting resulted in a significant increase in γ-H 2 AX foci per cell, whereas dCas9 with sgRNA did not. Statistical significance determined by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: After another centrifugation, the supernatant was removed and cells were resuspended in 500 μL of 1× Annexin V Binding Buffer (from Annexin V-FITC/PI Apoptosis Kit, Elabscience E-CK-A211).

Techniques: CRISPR, Flow Cytometry, Electroporation, Immunofluorescence

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: PGCs are more sensitive to DNA double-strand breaks than somatic cells. (A) Cell viability curves after 24 h treatment with increasing concentrations of etoposide (ETP; x-axis shows log 10 [ETP]). PGC viability dropped sharply even at low ETP doses, whereas CEF cells were more tolerant (viability data are mean ± SD of triplicates). (B) Apoptosis detection in PGCs treated with low-dose ETP. The proportion of Annexin V + /PI + cells (late apoptosis) was significantly elevated even at 0.03 µM ETP. (C) Western blot analysis of γ-H 2 AX protein in PGCs treated with varying ETP concentrations (left panel; β-actin as loading control). The bar graph (right) shows the ratio of γ-H 2 AX to β-actin band intensity, with a marked increase at 3 µM ETP (p-values are indicated in the figure). (D) Quantification of γ-H 2 AX foci per cell in THP-1 cells, male PGCs, and female PGCs after exposure to X-ray doses of 0, 2, 4, 6, and 8 Gy. After 48 h recovery, γ-H 2 AX foci increased significantly with higher radiation in THP-1, male PGCs, and female PGCs. Female PGC data points are red squares; male PGCs are blue squares; THP-1 are black circles. (E) Cell-cycle distribution of male vs. female PGCs after DNA damage. PGCs were irradiated (2, 4, 6 Gy), cultured 48 h, and analyzed by flow cytometry for cell-cycle phase (propidium iodide staining). Stacked bars show the percentage of cells in G 0 G 1 , S, and G 2 /M phases in untreated vs. irradiated cells. After damage, female PGCs predominantly arrested in G 2 /M (increased G 2 fraction), whereas male PGCs accumulated in S phase. Statistical significance by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: After another centrifugation, the supernatant was removed and cells were resuspended in 500 μL of 1× Annexin V Binding Buffer (from Annexin V-FITC/PI Apoptosis Kit, Elabscience E-CK-A211).

Techniques: Western Blot, Control, Irradiation, Cell Culture, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Clinical-grade cryopreservation unlocks transplant-ready human pancreatic and stem cell–derived islets for diabetes therapy

doi: 10.64898/2026.04.25.720819

Figure Lengend Snippet: ( A, B ) Bulk RNA-seq volcano plots comparing fresh versus VR native islets ( A ) and SC-islets ( B ). ( C ) Heatmap of differentially expressed genes in VR versus control SC-islets. ( D, E ) Pathway analyses highlighting enrichment of apoptosis, cytokine signaling, and stress responses in SC-islets (PANTHER categories; box-and-whisker plots). ( F ) Confocal images of Annexin V staining in VR islets and SC-islets, indicating apoptosis. Scale bar = 75 µm. ( G ) qPCR analysis of apoptosis-related gene expression in VR-treated SC-islets (mean log₂ fold change [log₂FC], n = 3–4 per group; Student’s t -test, * = p < 0.05). Abbreviations: FDR, false discovery rate; HSR, heat shock response; ISR, integrated stress response; OSR, oxidative stress response; SC, stem cell; UPR, unfolded protein response; VR, vitrified and rewarmed.

Article Snippet: Then, 5× Annexin V binding buffer (Cat. #99902, Biotium) was diluted in deionized water to prepare 1× binding buffer.

Techniques: RNA Sequencing, Control, Whisker Assay, Staining, Gene Expression