Journal: MethodsX

Article Title: Feline leukocyte immunophenotyping: an optimised whole-blood flow cytometry protocol

doi: 10.1016/j.mex.2026.103869

Figure Lengend Snippet: Representative leukocyte-gated histograms illustrating antibody titration for feline leukocyte immunophenotyping. All histograms display singlet leukocytes, defined by FSC-A versus SSC-A morphological gating, followed by FSC—H versus FSC-A singlet discrimination. Titration was performed for CD18, CD21, CD45R, CD4, CD5 and CD8 monoclonal antibodies using three antibody volumes: 10 µL (1:10 dilution), 5.0 µL (1:20 dilution), 3.0 µL (1:33 dilution) and 1.5 µL (1:66 dilution). Minimal working volumes were selected based on optimal signal-to-noise ratios.

Article Snippet: Step 2 – Leukocytes extracellular staining Materials • EDTA Whole blood sample ± Transfix® • 200 μl pipettes • 100 μl pipettes • 10 μl pipettes • Flow cytometry tubes • Permanent marker • Cytometer tube rack Reagents • Monoclonal antibodies: ○ CD5 Anti-cat – clone FE1.1B11 (BIO-RAD®) ○ CD4 Anti-cat – clone vpg34 (BIO-RAD®) ○ CD8 Anti-cat alpha/beta purified – clone vpg9 (BIO-RAD®) ○ Rat Anti-Mouse IgG1 – clone X56 (BIO-RAD®) ○ CD18 Mouse Anti-Dog – clone CA1.4E9 (BIO-RAD®) ○ CD21 Mouse Anti-Dog – clone CA2.1D6 (BIO-RAD®) ○ CD45R Rat Anti-Mouse – clone RA3–6B2 (BIO-RAD®) • 10x Red blood cells (RBC) lysis buffer solution (BD FACSTM lysing solution) • PBS 1% solution Equipment • Countess TM 3 (Thermo Fisher Scientific, USA) • Freezer • Dark incubation chamber • Timer • Vortex (MX-S®, China) • Centrifuge (model 5810R, Eppendorf®, Germany) • Flow cytometry analyser BD FACSCanto II (Becton Dickinson (BD), San Jose, USA) Methods 1.

Techniques: Titration, Bioprocessing

Journal: bioRxiv

Article Title: Inflammasome activation drives gasdermin-independent plasma membrane rupture by clustering ninjurin-1 in macrophages

doi: 10.64898/2026.04.10.717393

Figure Lengend Snippet: (A) A schematic model of DmrBASC fusion protein. (B) The design of tandem gRNA targeting GSDMD and GSDME. (C) Protein from DmrBASC – Gsdmd/e DKO iBMDM were analyzed by western blot. (D) DmrBASC – Gsdmd/e DKO iBMDM were pretreated with VX-765 for 30 min and treated with 100 nM B/B dimerizer for 4 h. The levels of LDH in the supernatants were assessed. (E and F) DmrBASC – Gsdmd/e DKO iBMDM were transduced with lentiviral vector encoding Kusabira Orange and images were captured by confocal microscopy in the presence of SYTOX Green. (E) Montage images and (F) quantification of fluorescent signals. (G–I) DmrBASC –iBMDM were pre-treated with 5 mM glycine and treated with 100 nM B/B dimerizer in the presence (G and H) or absence of SYTOX Green (I). (G and H) Images were visualized by confocal microscopy. (H) The percentage of SYTOX Green-positive cells was quantified. (I) The levels of LDH were assessed at 4 h. (J and K) DmrBASC – Gsdmd/e DKO iBMDM were transduced with LentiCRISPRv2 expressing GFP or Ninj1 -targeted gRNA. (J) NINJ1 protein expression was analyzed by western blot. (K) Cells were treated with100 nM B/B dimerizer for 4 h and stained with FITC-Annexin V and 7-AAD for flowcytometric analysis. (D, G, H, I, and K) The data were obtained from 3 independent experiments. Each dot represents independent experiments; bar indicates mean ± SD. (E and F) The data were obtained from 2 independent timelapse imaging. Light orange lines and light green lines indicate individual cells, and the deep orange line and deep green line indicates the mean of cells. Statistical significance was calculated using two-way ANOVA with Tukey’s post hoc test. ** p < 0.01, **** p < 0.0001.

Article Snippet: The cells were labeled with the following antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD45R (11-0452, eBioscience, San Diego, CA), phycoerythrin (PE)-conjugated anti-Ly6G (561104, BD Biosciences), and allophycocyanin (APC)-conjugated anti-CD45 (17-0451, eBioscience).

Techniques: Western Blot, Transduction, Plasmid Preparation, Confocal Microscopy, Expressing, Staining, Imaging

Journal: bioRxiv

Article Title: Inflammasome activation drives gasdermin-independent plasma membrane rupture by clustering ninjurin-1 in macrophages

doi: 10.64898/2026.04.10.717393

Figure Lengend Snippet: (A–F) Primary peritoneal macrophages isolated from Gsdmd –/– Gsdme –/– mice were primed with Pam3CSK4 (100 ng/mL) for overnight and treated with 5 µM nigericin in the presence of FITC-Annexin V and SYTOX Deep red. Z-stack time-lapse imaging was performed at 5 min-intervals. Annexin V-positive cells were tracked and analyzed. T 0 is defined as a time frame in which FITC-Annexin V signals were detected. (A and B) Representative cell in which exposure of PtdSer precedes the plasma membrane permeabilization. (A) Montage images and (B) quantification of fluorescent signals. (C and D) Representative cell in which exposure of PtdSer coincides with plasma membrane permeabilization. (C) Montage images and (D) quantification of fluorescent signals. (E and F) Representative plots of SYTOX Deep red (E) and FITC-Annexin V (F). Gray lines indicate individual cells, and red line (E) or green line (F) indicates the median of all cells obtained from a single mouse. (G–I) DmrBASC-Gsdmd/e DKO iBMDM were treated with100 nM B/B dimerizer for 4 h. Cells were stained with FITC-Annexin V and 7-AAD and analyzed by flow cytometry. (G) Representative plot, (H) the percentage of each population, and (I) the percentage of Annexin V + 7-AAD + cells. Each dot represents independent experiments; the bar indicates mean ± SD. (J–O) Primed Gsdmd –/– Gsdme –/– macrophages were stained with 5 µM CFSE and then treated with 5 µM nigericin in the presence of SYTOX Deep red. Z-stack time-lapse imaging was performed at 30-min intervals. (J and K) Representative 3D images of cells (J) before stimulation and 60 min after nigericin treatment (K). (L–O) The cell volume and sphericity in live cells were tracked. Representative plots of volume (L) and sphericity (N). Gray lines indicate individual cells, and the red line indicates the mean of all cells obtained from a single mouse. (M and O) Each dot represents the mean values of cells derived from individual mice. Cell volume and sphericity were assessed at one frame before the first detection of SYTOX Deep red-positive cells. (A–F, J–O) The data were obtained from cells derived from 6 mice. (G–I) The data are obtained from 3 independent experiments. Statistical significance was calculated using Student’s t test. * p < 0.05

Article Snippet: The cells were labeled with the following antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD45R (11-0452, eBioscience, San Diego, CA), phycoerythrin (PE)-conjugated anti-Ly6G (561104, BD Biosciences), and allophycocyanin (APC)-conjugated anti-CD45 (17-0451, eBioscience).

Techniques: Isolation, Imaging, Clinical Proteomics, Membrane, Staining, Flow Cytometry, Derivative Assay

Journal: bioRxiv

Article Title: Inflammasome activation drives gasdermin-independent plasma membrane rupture by clustering ninjurin-1 in macrophages

doi: 10.64898/2026.04.10.717393

Figure Lengend Snippet: (A and B) Primary peritoneal macrophages isolated from Gsdmd –/– Gsdme –/– mice were primed with Pam3CSK4 (100 ng/mL) for overnight and treated with 5 µM nigericin in the presence of FITC-Annexin V and SYTOX Deep red. Z-stack timelapse imaging was performed at 5 min interval. Annexin V-positive cells were tracked and analyzed. T 0 is defined as a time frame in which FITC-Annexin V signals was detected. Quantification of fluorescent signals of Annexin V (A) and SYTOX Deep red (B). Each dot represents medians of all cells obtained from single mouse (N=6). (C–I) DmrBASC – Gsdmd/e DKO iBMDM were stained with 5 µM CFSE and treated with100 nM B/B dimerizer for 30 min. Z-stack imaging was performed. (C and D) Representative 3D images of unstimulated cells (C) and cells (D) treated with B/B dimerizer. The cell volume (D and E), sphericity (F and G), and surface area (H and I) were analyzed. (D, F, and H) Each dot represents an individual cell from three independent experiments. (E, G, I) Each dot represents mean value per experiment. (J and K) Primary peritoneal macrophages isolated from Gsdmd –/– Gsdme –/– mice were primed with Pam3CSK4 (100 ng/mL) for overnight and labeled with Flipper-TR for 15 min. The labeled cells were pretreated with 5 mM glycine and stimulated with 5 µM nigericin for 30 min. Fluorescent lifetime was measured using FLIM. (J) Representative FLIM imaging and (K) lifetime of Flipper-TR. Each dot represents individual cell.

Article Snippet: The cells were labeled with the following antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD45R (11-0452, eBioscience, San Diego, CA), phycoerythrin (PE)-conjugated anti-Ly6G (561104, BD Biosciences), and allophycocyanin (APC)-conjugated anti-CD45 (17-0451, eBioscience).

Techniques: Isolation, Imaging, Staining, Labeling

Journal: bioRxiv

Article Title: Inflammasome activation drives gasdermin-independent plasma membrane rupture by clustering ninjurin-1 in macrophages

doi: 10.64898/2026.04.10.717393

Figure Lengend Snippet: (A–F) DmrBASC-Gsdmd/e DKO iBMDM were transduced with LentiCRISPRv2 expressing GFP, Xkr8, Ano6, and Tmem63b-targeted gRNA. The cells were treated with 100 nM B/B dimerizer. (A and B) Cells were stimulated in the presence of SYTOX Green. (A) Images were visualized by confocal microscopy at 2h. (B) The percentage of SYTOX Green-positive cells was quantified. (C) The levels of LDH were assessed at 4 h. (D–F) Cells were stimulated in the presence of FITC-Annexin V and SYTOX Deep red for 2 h and analyzed by high content analysis. (D) Representative histogram of Annexin V intensity. (E) The ratio of Annexin V+ cells and Annexin V+ SYTOX Deep red+ cells was quantified. (A–F) The data were obtained from 3 (A, B, D–F) or 5 (C) independent experiments. Each dot represents individual experiments; the bar indicates mean ± SD. Statistical significance was calculated using two-way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The cells were labeled with the following antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD45R (11-0452, eBioscience, San Diego, CA), phycoerythrin (PE)-conjugated anti-Ly6G (561104, BD Biosciences), and allophycocyanin (APC)-conjugated anti-CD45 (17-0451, eBioscience).

Techniques: Transduction, Expressing, Confocal Microscopy, High Content Screening