flow cytometry staining buffer  (Thermo Fisher)

Journal: STAR Protocols

Article Title: Protocol for isolating and culturing microglia from the adult mouse brain using a magnetic-activated cell sorting system

doi: 10.1016/j.xpro.2026.104471

Figure Lengend Snippet: Flow cytometry for MACS-isolated microglia purity (A and B) Total CD11b-positive cells from MACS columns; (C) Cell viability assessed by Zombie Red staining; (D) Infiltrating leukocytes, including neutrophils (Ly6G+) and T lymphocytes (CD3+); (E) Proportions of microglia (CD11b+CD45low) versus monocytes/border-associated macrophages (CD11b+CD45high); (F) Proportion of resting (homeostatic) microglia identified as CD11b+TMEM119+ cells.

Article Snippet: Note: We use commercial flow cytometry staining buffer from eBioscience (Cat# 00-4222-26), which is PBS-based formulation designed to prevent non-specific antibody binding and maintain cell stability during flow cytometry.

Techniques: Flow Cytometry, Isolation, Staining

Journal: bioRxiv

Article Title: NFATC2 in pancreatic cancer-associated fibroblasts predicts treatment response and facilitates ERBB-targeted therapies

doi: 10.64898/2026.04.04.716465

Figure Lengend Snippet: (A) Western blot showing ectopic NFATC2 expression in pancreatic CAFs (pCAF) generated using lentiviral transduction. GAPDH served as loading control. WT, wild-type pCAFs; NFATC2+, NFATC2-expressing pCAFs. Uncropped blots are shown in Supplementary Figure 3A. (B) Representative fluorescence microscopy images of PANC1-pCAF co-cultures. PANC1 cells are labeled with mCherry (red) and pCAF cells with EGFP (green). WT and NFATC2+ pCAF co-cultures are shown on days 1-3 under vehicle or FOLFIRINOX treatment. Scale bar, 10μm. (C) Quantification of PANC1 cell numbers using CellProfiler from fluorescence images showing that NFATC2+ CAFs significantly reduce cancer cell proliferation, particularly under FOLFIRINOX treatment. Data are presented as mean ± SEM (n=3 independent experiments). Upper panel: p<0.0001. Lower panel: p=0.0092, two-way ANOVA. (D) Flow cytometry analysis of PANC1-pCAF co-culture systems with WT pCAFs (left) and NFATC2+ pCAF (right). Scatter plots display mCherry fluorescence (x-axis) versus eGFP fluorescence (y-axis), PANC1 cells (mCherry+, eGFP-) seen in the lower right quadrant. (E) Annexin V-FITC apoptosis assay by flow cytometry. Density plots showing the fraction of apoptotic cells (Q2, top-right quadrant) relative to live cancer cells (Q3, bottom-right quadrant) following co-culture with WT or NFATC2+ pCAFs ± FOLFIRINOX. In FOLFIRINOX-treated cells, the apoptotic fraction increased from 19.1% in WT pCAF co-cultures to 33.0% in NFATC2+ pCAF co-cultures, indicating enhanced chemotherapy-induced apoptosis in cancer cells co-cultured with NFATC2+ pCAFs.

Article Snippet: Cell pellets were resuspended in 1 mL flow cytometry buffer (Invitrogen, #00-4222-26) and passed through 40 μm cell strainers (Corning, #CLS431750) to remove cell aggregates and debris.

Techniques: Western Blot, Expressing, Generated, Transduction, Control, Fluorescence, Microscopy, Labeling, Flow Cytometry, Co-Culture Assay, Apoptosis Assay, Cell Culture

Journal: bioRxiv

Article Title: NFATC2 in pancreatic cancer-associated fibroblasts predicts treatment response and facilitates ERBB-targeted therapies

doi: 10.64898/2026.04.04.716465

Figure Lengend Snippet: (A) Representative fluorescence microscopy images of PANC1-pCAF co-culture system under different treatment conditions. Cancer cells (mCherry+, red) and CAFs (eGFP+, green) were treated with vehicle control, standard chemotherapy regimens (FOLFIRINOX or Gemcitabine plus Paclitaxel) alone, or chemotherapy combined with ERBB-targeted inhibitors including Trastuzumab, Erlotinib and Neratinib. Scale bar, 10μm. (B) Quantification of PANC1 cancer cell numbers from fluorescence microscopy analysis. Data represent mean ± SEM from three independent experiments. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, t-test. (C) Normalized cell numbers of PANC1 from fluorescence microscopy analysis based on vehicle control from each group. Data represent mean ± SEM from three independent experiments. Same statistical significance marks used as (B). (D) Flow cytometry analysis of the co-culture system after three-day treatment. Density plots display mCherry fluorescence (x-axis) versus eGFP fluorescence (y-axis), revealing distinct cell populations: PANC1 cells (mCherry+, eGFP-, lower right quadrant), CAFs (mCherry-, eGFP+, upper left quadrant), and cellular debris (lower left quadrant). Quantification of PANC1 cell proportions demonstrating a marked reduction in the cancer cell population following chemotherapy combined with ERBB-targeted inhibitors, particularly Trastuzumab and Neratinib, in the presence of NFATC2+ pCAFs.

Article Snippet: Cell pellets were resuspended in 1 mL flow cytometry buffer (Invitrogen, #00-4222-26) and passed through 40 μm cell strainers (Corning, #CLS431750) to remove cell aggregates and debris.

Techniques: Fluorescence, Microscopy, Co-Culture Assay, Control, Flow Cytometry