intracellular flow cytometry kit 13593s  (Cell Signaling Technology Inc)

Journal: Materials Today Bio

Article Title: A microfluidic tumor-on-chip platform deciphers hypoxia-driven FOXO3a/PD-L1 signaling in gastric cancer immunotherapy resistance

doi: 10.1016/j.mtbio.2025.101925

Figure Lengend Snippet: Microfluidic Tumor-on-chip model. a Overall schematic diagram (top left) and physical image (bottom left)image of the microfluidic tumor-on-chip model. Layer-by-layer schematic diagrams of chip were shown right. b Schematic diagram of the chamber layer. In the the microchamber, GC cells were embedded within extracellular matrix gel while HUVECs were arranged at the opening of the microchamber. CD8 + T cells were perfused with anti-PD-1 antibody in the microchannels. c Confocal images showing the spatial distribution of the cells inside the tumor-on-chip model. AGS cells (green) were embedded within the extracellular matrix gel, while CD8 + T cells (blue) and HUVEC cells (red) were encapsulated within the microchannels. d The proliferative capacity of GC cells in the tumor-on-chip model and traditional 96-well plate at 1 day, 7 days, and 14 days, as assessed using CCK-8 assays. e and f Flow cytometry analysis of the cell apoptosis rate in GC cells grown on the tumor-on-chip model and traditional 96-well plates at 0 days, 7 days, and 14 days. Values represent the mean ± SD. ∗ p < 0.05; ∗∗∗∗ p < 0.0001; ns, no significance. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Flow cytometry analysis of intracellular proteins in tumor cells of syngeneic mouse model was determined using an Intracellular Flow Cytometry Kit (Cell Signaling Technology, Danvers, MA, USA; 13593S).

Techniques: CCK-8 Assay, Flow Cytometry

Journal: Materials Today Bio

Article Title: A microfluidic tumor-on-chip platform deciphers hypoxia-driven FOXO3a/PD-L1 signaling in gastric cancer immunotherapy resistance

doi: 10.1016/j.mtbio.2025.101925

Figure Lengend Snippet: Hypoxia induced the expression of immunosuppressive genes in GC cells. a Schematic diagram of GC cell separation. After culturing GC cells in the matrix gel for 7 days, the gel was degraded, and the separation was conducted via FACS. b Flow cytometry analysis showing two distinct clusters of GC cells and CD8 + T cells before sorting. Following sorting, only AGS cells were present. c KEGG enrichment analysis indicating the pathways enriched for DEGs in AGS cells under a hypoxic TME. d Heatmap of 799 DEGs (fold change ≥1.5 and P value <0.05 in any pairwise comparison) under hypoxia versus normoxic environments. e Correlation analysis revealing the interrelationships among various DEGs in AGS cells under a hypoxic TME. f Venn diagram illustrating the overlapping genes within the signaling pathways of AGS cells under a hypoxic TME. g Experssion level of FOXO3A , HIF1A , and PDL1 in MKN-45. h Comparison of gene expression in AGS cells cultured in traditional 96-well plate versus the tumor-on-chip model, suggesting cellular exhaustion in the traditional 96-well plate 3D culture method. Values represent the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no significance.

Article Snippet: Flow cytometry analysis of intracellular proteins in tumor cells of syngeneic mouse model was determined using an Intracellular Flow Cytometry Kit (Cell Signaling Technology, Danvers, MA, USA; 13593S).

Techniques: Expressing, Flow Cytometry, Comparison, Protein-Protein interactions, Gene Expression, Cell Culture

Journal: Materials Today Bio

Article Title: A microfluidic tumor-on-chip platform deciphers hypoxia-driven FOXO3a/PD-L1 signaling in gastric cancer immunotherapy resistance

doi: 10.1016/j.mtbio.2025.101925

Figure Lengend Snippet: High expression of FOXO3a induced resistance to immunotherapy in vivo. a-c Six-week-old BALB/c mice were injected subcutaneously with MFC, sh-FOXO3a or sh-control MFC cells (1 × 10 7 cells). Endpoint images of syngeneic mouse tumor formed by MFC cells in BALB/c mice ( a ). Volumes, weights ( b ), and changes in volume every 2–3 days ( c ) were recorded (n = 6 for each group). d IHC analysis was performed to determine the density of CD3 + , CD4 + , and CD8 + cells, as well as PD-L1 expression in the subcutaneous syngeneic mouse tumors. Scale bar = 100 μm (left) and 50 μm (right). e Proportions of IFN + cells among tumor-infiltrating NK and CD8 + T cells, as determined using flow cytometry. Simultaneously, the prevalence of PD-L1 + and PD-L2 + cells within the TME was examined. f Expression levels of HIF-1α, PD-L1 and FOXO3a in GC patient tissues before anti-PD-1 treatment were evaluated by IF staining ( n = 15 PR patients; n = 15 PD patients). Scale bar = 50 μm g The overall survival of patients with GC with high and low expression levels of FOXO3a who underwent anti-PD-1 antibody treatment was investigated. Values represent the mean ± SD. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: Flow cytometry analysis of intracellular proteins in tumor cells of syngeneic mouse model was determined using an Intracellular Flow Cytometry Kit (Cell Signaling Technology, Danvers, MA, USA; 13593S).

Techniques: Expressing, In Vivo, Injection, Control, Flow Cytometry, Staining

Journal: Genes to Cells

Article Title: Establishment of a Novel Caco‐2‐Based Cell Culture System for Human Sapovirus Propagation

doi: 10.1111/gtc.70007

Figure Lengend Snippet: Susceptibility of various cell lines to HuSaV infection. (A) Changes in HuSaV RNA copy numbers in the culture supernatants of HuTu80 and HEK293T cells following inoculation with a HuSaV GI.1 (AH20)‐positive stool suspension (~4 × 10 7 copies/well in 96‐well plates). Each dot represents individual data points; bars indicate the geometric mean value of the HuSaV RNA copy numbers; error bars denote the geometric standard deviation (SD). This experiment was performed once with five technical replicates. (B) Changes in HuSaV RNA copy numbers in the culture supernatants of Caco‐2, HCT15, HCT116, Caco‐2/Cas9, and C2BBe1 cells following inoculation with a HuSaV GI.1 (AH20)‐positive stool suspension (~2 × 10 6 copies/well in 96‐well plates). Each dot represents individual data points; bars indicate the geometric mean HuSaV RNA copy numbers; error bars denote the geometric SD. This experiment was performed once with five technical replicates. (C) Immunofluorescence staining of the viral protein VP1 in HuTu80, HEK293T, Caco‐2, and Caco‐2/Cas9 cells at 3 dpi with a HuSaV GI.1 (AH20)‐positive stool suspension; upper panels: 4× objective lens, lower panels: 40× objective lens. (D) Flow cytometry analysis of Caco‐2 and Caco‐2/Cas9 cells infected with a HuSaV GI.1 (AH20)‐positive stool suspension at 4 dpi. Blue: VP1‐negative cells (uninfected), red: VP1‐positive cells (infected).

Article Snippet: The cells were pelleted by centrifugation, fixed with 4% formaldehyde for 15 min, and permeabilized with methanol for 10 min at 4°C using the Intra Cellular Flow Cytometry kit (Cell Signaling Technology, MA, USA).

Techniques: Infection, Suspension, Standard Deviation, Immunofluorescence, Staining, Flow Cytometry

Journal: Genes to Cells

Article Title: Establishment of a Novel Caco‐2‐Based Cell Culture System for Human Sapovirus Propagation

doi: 10.1111/gtc.70007

Figure Lengend Snippet: Immunofluorescence and flow cytometry analysis of highly HuSaV‐susceptible Caco‐2MC cells. (A) Immunofluorescence staining at 4 dpi with HuSaV GI.1 (AH20) on cloned cells derived from Caco‐2/Cas9 cells; (left) clone M (Caco‐2M), (right) clone P (Caco‐2P); blue: Nuclei (Hoechst). (B) Flow cytometry at 4 dpi following infection with HuSaV GI.1 (AH20) in Caco‐2M, Caco‐2MC, and Caco‐2ME cells; blue: VP1‐negative cells (uninfected), red: VP1‐positive cells (infected). (C) Immunofluorescence staining of Caco‐2MC cells at 4 dpi following infection with HuSaV GI.1 (AH20). (D) Flow cytometry at 4 dpi following infection with HuSaV GI.1 (AH20) in Caco‐2PG cells.

Article Snippet: The cells were pelleted by centrifugation, fixed with 4% formaldehyde for 15 min, and permeabilized with methanol for 10 min at 4°C using the Intra Cellular Flow Cytometry kit (Cell Signaling Technology, MA, USA).

Techniques: Immunofluorescence, Flow Cytometry, Staining, Clone Assay, Derivative Assay, Infection

Journal: Parasitology Research

Article Title: Tick salivary cystatin Iristatin limits the virus replication in skin of tick-borne encephalitis virus–infected mice

doi: 10.1007/s00436-024-08441-5

Figure Lengend Snippet: Iristatin interferes with signaling pathways activation and exerts anti-apoptotic effect in TBEV-infected dendritic cells. DC were activated by imiquimod (IQ; 2 µg/ml) for 3 h in the presence or absence of Iristatin (3 µM) and protein cell lysates were analyzed for the activation of signaling pathways using PathScan intracellular signaling array ( a ). DC were infected by Hypr at MOI 5 for indicated times in the presence or absence of Iristatin (3 µM) and then Erk1/2 phosphorylation was analyzed by immunoblotting. Membranes were re-probed to determine the level of total Erk1/2 proteins. Proteins were visualized by chemiluminescence and representative blot with relative phosphorylation is shown ( b ). DC were non-infected or infected by Hypr at MOI 5 in the presence or absence of Iristatin (6 µM) and the percentage of active caspase-3 positive cells was measured by flow cytometry ( c ). * p ≤ 0.05; **** p ≤ 0.0001 ns = not significant

Article Snippet: After 3 h, the cell protein lysates were prepared and analyzed using the PathScan® intracellular signaling array kit (#7323, Cell Signaling Technology) according to the manufacturer’s instruction.

Techniques: Protein-Protein interactions, Activation Assay, Infection, Phospho-proteomics, Western Blot, Flow Cytometry

Journal: mBio

Article Title: SIRT7 remodels the cytoskeleton via RAC1 to enhance host resistance to Mycobacterium tuberculosis

doi: 10.1128/mbio.00756-24

Figure Lengend Snippet: SIRT7 deficiency impairs macrophage phagocytosis and bactericidal capacity. ( A and B ) Flow cytometry analyses and quantification of GFP-H37Ra phagocytosis by Sirt7 −/− BMDMs compared with Sirt7 +/+ BMDMs. Histograms illustrate the percentage of macrophages that phagocytosed GFP-H37Ra, with GFP + BMDMs indicating cells that ingested GFP-H37Ra. ( C, D, J, and K ) Intracellular CFU counts of H37Ra or H37Rv assessed 4 h ( C and D ) or 72 hours ( J and K ) post-infection in Sirt7 −/− BMDMs and Sirt7 +/+ BMDMs. (E through I) Comparative analysis of the percentage of Sirt7 −/− and Sirt7 +/+ BMDMs phagocytosing apoptotic bodies, beads, Staphylococcus aureus , Escherichia coli , and Salmonella Typhi. After phagocytosis, BMDMs engulfing beads can be detected by APC fluorescence, while the ingestion of pHrodo red-labeled apoptotic bodies and bacteria can be detected by PE fluorescence. ( L and M ) The survival ratio of H37Ra or H37Rv was evaluated in Sirt7 −/− BMDMs compared to WT controls. Data are presented as means ± SEM, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, as determined by Student’s two-tailed unpaired t-test. Each experiment was independently replicated three times.

Article Snippet: The procedure adhered to the steps outlined in the Mouse Intracellular (nuclear/transcription factor) Protein Flow Cytometry Workflow Kit (Invitrogen, # A53017).

Techniques: Flow Cytometry, Infection, Fluorescence, Labeling, Bacteria, Two Tailed Test

Journal: mBio

Article Title: SIRT7 remodels the cytoskeleton via RAC1 to enhance host resistance to Mycobacterium tuberculosis

doi: 10.1128/mbio.00756-24

Figure Lengend Snippet: SIRT7 induces cytoskeletal remodeling via RAC1. ( A ) RAC1activation in H37Rv-infected Sirt7 −/− and Sirt7 +/+ BMDMs assayed at 0 min and 30 min post-infection. “Input” represents RAC1 protein levels in whole-cell lysates, while “IP: PAK1-PBD” indicates active RAC1 levels. ( B ) Immunoblot and immunoprecipitation assays illustrating the interaction of SIRT7 and RAC1 in THP1-derived macrophages transfected with si- RAC1 or normal control (si-NC) siRNA. ( C and G ) Flow cytometry analysis quantifying the phagocytosis of GFP-H37Ra by Sirt7 −/− and Sirt7 +/+ BMDMs under the conditions of RAC1 inhibitor or activator treatment. GFP + BMDMs represent cells that phagocytosed GFP-H37Ra. ( D ) Intracellular CFU counts of H37Rv at 4 hours post-infection in Sirt7 −/− and Sirt7 +/+ BMDMs. ( E and H ) Following a 4-hour infection period with H37Rv, macrophages were washed with phosphate-buffered saline (PBS) to eliminate unphagocytosed bacteria. Subsequently, a fresh complete medium was introduced, either containing an RAC1 inhibitor or activator. CFU counts were determined 68 hours later. ( F and I ) The survival ratio of H37Rv was evaluated in Sirt7 −/− BMDMs compared to WT BMDMs, following treatment with either RAC1 inhibitor or activator. ( J and K ) Immunoblot analysis was performed to assess the expression of LC3II in Sirt7 −/− and Sirt7 +/+ BMDMs treated with an RAC1 inhibitor or Baf-A1. The expression levels of LC3II were quantified and are shown relative to GAPDH, as indicated below the corresponding bands. Data are presented as means ± SEM, * P < 0.05, ** P < 0.01, and *** P < 0.001, as determined by one-way ANOVA with Tukey’s multiple comparisons test. Each experiment was independently replicated two to three times.

Article Snippet: The procedure adhered to the steps outlined in the Mouse Intracellular (nuclear/transcription factor) Protein Flow Cytometry Workflow Kit (Invitrogen, # A53017).

Techniques: Infection, Western Blot, Immunoprecipitation, Derivative Assay, Transfection, Control, Flow Cytometry, Saline, Bacteria, Expressing

Journal: mBio

Article Title: SIRT7 remodels the cytoskeleton via RAC1 to enhance host resistance to Mycobacterium tuberculosis

doi: 10.1128/mbio.00756-24

Figure Lengend Snippet: Overexpression of Sirt7 enhanced host anti-TB immunity. ( A and B ) Intracellular CFU counts of H37Rv at 4 hours and 72 hours post-infection in Sirt7 +/+ and Sirt7 TG BMDMs. ( C ) The survival ratio of H37Rv was evaluated in Sirt7 TG BMDMs compared to WT controls. ( D ) LC3II expression was elevated in Sirt7 TG BMDMs compared to WT controls. ( E ) RAC1 activation in H37Rv-infected Sirt7 +/+ and Sirt7 TG BMDMs assayed at 0 min and 30 min post-infection, with “Input” indicating total RAC1 and “IP: PAK1-PBD” indicates active RAC1. ( F and G ) Bacterial burden in the lungs and spleens of Sirt7 +/+ and Sirt7 TG mice was assessed in the tissue homogenates. ( H ) Histopathological analysis of lung sections from Sirt7 +/+ and Sirt7 TG mice infected with H37Rv. Sections were stained with hematoxylin and eosin, and images were captured using a NanoZoomer digital pathology system. Data are presented as means ± SEM, * P < 0.05, ** P < 0.01, and *** P < 0.001 by Student’s two-tailed unpaired t-test. Each experiment was independently replicated two to three times.

Article Snippet: The procedure adhered to the steps outlined in the Mouse Intracellular (nuclear/transcription factor) Protein Flow Cytometry Workflow Kit (Invitrogen, # A53017).

Techniques: Over Expression, Infection, Expressing, Activation Assay, Staining, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: AFM41a: A Novel PAD2 Inhibitor for Sepsis Treatment—Efficacy and Mechanism

doi: 10.7150/ijbs.97166

Figure Lengend Snippet: Pad2 deficiency promoted Alveolar macrophages (AMs) towards M2 polarization and increased phagocytosis. The Pad2 -/- and WT mice were infected with 2.5 X 10 6 CFU Pseudomonas aeruginosa (PA) 19660/mouse for 24 hours. (A) AMs from Pad2 -/- and WT mice were lysed, and the expression of classical cytokine genes ( Tnfα, Il6, and Il10 ) and macrophage polarization signature genes ( Nos2, Ccl2, Mrc1, and Arg1 ) was quantified by qRT-PCR. (B) BALF was collected from both groups of mice, and the levels of inflammation cytokines (TNF-α, IL-6, and IL-10) were determined using ELISA. (C) AMs were isolated from Pad2 -/- and WT mice, cell lysates from AMs were analyzed by western blotting to assess the protein levels of macrophage polarization markers, iNOS, and Arg-1. (D) AMs were subjected to immunofluorescence staining to evaluate the expression of macrophage polarization markers, iNOS, and Arg-1. (E) Isolated AMs from Pad2 -/- and WT mice were analyzed by flow cytometry to quantify the distribution of M1 and M2 macrophage populations. (F) Quantification of the proportion of M1 and M2 macrophages. (G) The phagocytic capacity of AMs was measured using pHrodo Red E. coli BioParticles. Data are representative of three independent experiments expressed as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.01.

Article Snippet: To investigate intracellular markers of macrophage activation, iNOS (17-5920-82, Invitrogen) and Arg-1 (IC5868P, R&D) were stained using the Staining Intracellular Antigens for Flow Cytometry kit (00-5521-00, Thermo Fisher Scientific), following the user guidelines provided by the company.

Techniques: Infection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Isolation, Western Blot, Immunofluorescence, Staining, Flow Cytometry