annexin binding buffer  (Fisher Scientific)

Journal: Science Advances

Article Title: Tumor gasdermin E acts as a rheostat to limit apoptotic cell survival and mutagenesis in anticancer immune environments

doi: 10.1126/sciadv.aeb1137

Figure Lengend Snippet: ( A ) Immunoblotting of SIINFEKL peptide–pulsed Neuro2a cell lines cocultured with activated OT-I T cells for 4 hours at ratios of T cells to target cells indicated. ( B ) Schematic representation of fluorescence-activated cell sorting (FACS) based on Caspase-3(DEVD)NucView and annexin V (AnnV) staining, applies to (C) to (E). Created in BioRender; B. Liu (2026), https://BioRender.com/y3nlv0g . ( C ) Rate of outgrowth from cells recovered from FACS sort, calculated on the basis of number of initially plated cells. Data were compiled from five independent experiments. Paired t test. ( D and E ) Quantification of nucleotide fragments with greater than three, four, five, or six single-nucleotide variants (SNVs) from various recovered populations as indicated. ( F ) The design of the ATM–fluorescence resonance energy transfer (FRET) reporter. Created in BioRender; B. Liu (2026), https://BioRender.com/jqdhdx7 . ( G ) Quantification of FRET loss signal from Neuro2a expressing the ATM-FRET reporter shown in (F). Neuro2a cells were SIINFEKL peptide pulsed and then coincubated with OT-I T cells for 6 hours at 10:1 T cell–to–target cell ratio. Data were pooled from three independent experiments. Unpaired t test. ( H ) In vivo growth of Neuro2a cells expressing the ATM-FRET sensor. ATM-FRET loss populations were sorted post BH3m treatment (1 μM ABT737 and 1 μM S63845 ). Cells were sorted for FRET-loss population at 2.5 hours, and 1 × 10 4 cells were injected into NSG mice ( n = 5 animals per group). Kaplan-Meyer survival curves. WT-base and GE KO–base refer to ATM-FRET–expressing cells that were not treated with BH3m. Log-rank (Mantel-Cox) test. * P < 0.05 and **** P < 0.0001. C3, active caspase-3.

Article Snippet: Cells were spun down and resuspended in DMEM supplemented with annexin binding buffer (99902, Biotium) and stained with annexin V-647 and/or Caspase-3(DEVD)NucView-488, as indicated in the text, for 10 min at 37°C.

Techniques: Western Blot, Fluorescence, FACS, Staining, Förster Resonance Energy Transfer, Expressing, In Vivo, Injection

Journal: Science Advances

Article Title: Tumor gasdermin E acts as a rheostat to limit apoptotic cell survival and mutagenesis in anticancer immune environments

doi: 10.1126/sciadv.aeb1137

Figure Lengend Snippet: ( A ) Coculture (1:1) of parental B16F10 cells expressing pBabe-Cerulean (blue) and B16F10 cells expressing GsdmD-C3 and mCherry (red) (see movie S1). ( B ) Times of individual cell shrinkage and cell membrane lysis, as in (A). Unpaired t test. ( C ) Apoptotic phase (shrinkage-lysis interval) from (B). One-way ANOVA. ( D ) Quantification of annexin V + SIINFEKL peptide–pulsed B16F10 [as in (B) and (C)] that remains fluorescent when coincubated with activated OT-I T cells at 4 hours. Two-way ANOVA. ( E ) Quantification of SIINFEKL peptide–pulsed B16F10 cell populations with ectopic caspase-3-activatable GSDMD (GD-C3) by fluorescence signal at 24 hours of coincubation with activated OT-I T cells. ( F ) As in (E) with Gsdme. [(E) and (F)] Paired t test. ( G ) Number of GCamp6s spikes observed from SIINFEKL peptide–pulsed Neuro2a cells, coincubated with activated OT-I T cells, 10:1 T cell–to–target cell ratio. Images were taken every 2 min for a duration of 3 hours immediately following the additional of OT-I T cells, and numbers of observable GCamp6s spikes were counted and plotted (see movies S2 to S4 and fig. S5C). Unpaired t test. ( H ) Design for (I) and (J). Created in BioRender; B. Liu (2026), https://BioRender.com/j67ipmw . ( I ) Bioluminescence signal intensity of Neuro2a in C57BL/6 animals ( n = 9 animals per group). Two-way ANOVA. ( J ) Kaplan-Meyer survival curves from C57BL/6 mice injected with Neuro2a cells ( n = 9 animals per group). ( K ) Design for (L). Created in BioRender; B. Liu (2026), https://BioRender.com/j67ipmw . ( L ) Bioluminescence signal intensity of Neuro2a in C57BL/6 animals (control, n = 9 animals; Gsdme −/− , n = 8 animals). ( M ) Kaplan-Meyer survival curves from C57BL/6 mice rechallenged with Neuro2a cells [ n = 5 animals per group; log-rank (Mantel-Cox) test]. ( N ) As in (M) with B16F10 [ n = 12 animals in naïve group, 14 animals in rechallenge group; log-rank (Mantel-Cox) test]. * P < 0.05, ** P < 0.01, and **** P < 0.0001. iv, intravenous; ip, intraperitoneal.

Article Snippet: Cells were spun down and resuspended in DMEM supplemented with annexin binding buffer (99902, Biotium) and stained with annexin V-647 and/or Caspase-3(DEVD)NucView-488, as indicated in the text, for 10 min at 37°C.

Techniques: Expressing, Membrane, Lysis, Fluorescence, Injection, Control

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

Journal: Veterinary Research

Article Title: Neu1 inhibition restrains BCoV replication and modulates ZBP1-dependent PANoptosis

doi: 10.1186/s13567-026-01729-7

Figure Lengend Snippet: BCoV infection induces the expression of PANoptosis-related genes in MDBK cells. A Western blot analysis of RIPK3, RIPK1, p-MLKL, and ZBP1 protein levels. B , C Relative mRNA levels of ZBP1 ( B ) and RIPK3 ( C ) determined by qPCR. D , E Protein ( D ) and mRNA ( E ) expression of CASP8. F Flow cytometric analysis of apoptosis via Annexin V-PE/7-AAD staining in control and BCoV-infected cells. G , H Protein ( G ) and mRNA ( H ) expression of GSDMD, CASP1, and ASC. I Relative mRNA expression of IL-1β . J , K Protein ( J ) and mRNA ( K ) expression of Neu1. L Cellular sialic acid levels, which decreased in a dose-dependent manner upon BCoV infection. Quantitative densitometry of the western blot results (normalized to β-actin/GAPDH) is shown in Additional file . The data are presented as the means ± SDs ( n = 3). Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The pellets were washed three times with 1× PBS and subsequently resuspended in 1× Annexin V-PE binding buffer (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCl 2 solution; Servicebio, Wuhan) at the appropriate cell density.

Techniques: Infection, Expressing, Western Blot, Staining, Control

Journal: Veterinary Research

Article Title: Neu1 inhibition restrains BCoV replication and modulates ZBP1-dependent PANoptosis

doi: 10.1186/s13567-026-01729-7

Figure Lengend Snippet: The interaction between Neu1 and ZBP1 regulates apoptosis and sialic acid levels in BCoV-infected MDBK cells. A , B Schematic representation and validation of stable MDBK Neu1kd and MDBK ZBP1kd cell lines. C Apoptosis analysis of WT, MDBK Neu1kd , and MDBK ZBP1kd cells following BCoV infection via Annexin V-PE/7-AAD flow cytometry. D , E Viral titers determined via the Reed–Muench method ( D ) and relative BCoV mRNA expression levels ( E ). F Immunofluorescence localization of Neu1 (red) and ZBP1 (green) in WT, MDBK Neu1kd , and MDBK ZBP1kd cells. Nuclei were stained with DAPI (blue), and merged signals (yellow) indicate colocalization. G Cellular sialic acid levels significantly decreased in both MDBK Neu1kd and MDBK ZBP1kd cells. H Immunofluorescence analysis further demonstrated that Neu1 or ZBP1 knockdown markedly reduced the fluorescence signal intensity during BCoV infection. The data are presented as the means ± SDs ( n = 3). The quantification of fluorescence intensity is shown in Additional file . Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The pellets were washed three times with 1× PBS and subsequently resuspended in 1× Annexin V-PE binding buffer (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCl 2 solution; Servicebio, Wuhan) at the appropriate cell density.

Techniques: Infection, Biomarker Discovery, Flow Cytometry, Endpoint Dilution Assay, Expressing, Immunofluorescence, Staining, Knockdown, Fluorescence