Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Investigation of in-vitro Anti-Cancer and Apoptotic Potential of Garlic-Derived Nanovesicles against Prostate and Cervical Cancer Cell Lines

doi: 10.31557/APJCP.2024.25.2.575

Figure Lengend Snippet: GDNVs Induce Apoptosis in Prostate and Cervical Cancer Cells. PI/annexin Vstaining and further analysis by flow cytometry dot plots in (a) HeLa and (b) PC-3 cells respectively showing induction of apoptosis after 48 hours. Data expressed as the mean ± S.D. Asterisk represents the statistically significant values in comparison to control (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: 400µl of binding buffer was added to each sample followed by the acquisition of the sample using flow cytometry FACS Calibur (BD Biosciences San Jose, CA).

Techniques: Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Stabilized gp120-specific CD4 for next-generation HIV-1 inhibitors

doi: 10.64898/2026.03.24.713825

Figure Lengend Snippet: a, Diagram of the flow cytometry experimental set-up. Mononuclear leukocytes were isolated by Lymphoprep gradient density separation, aliquoted, and stored frozen. Aliquots were thawed as needed and samples were prepared with live-dead staining and Human TruStain FcX Fc receptor blocking solution. The cells were then labeled with anti-CD19 conjugated to Brilliant Violet 650 and biotinylated CD4-Ig variants. After removing excess antibody, the cells were labeled with streptavidin conjugated to Alexa Fluor 488 and analyzed by flow cytometry. b-g, Flow cytometry plots of tonsillar cells. CD19 signal is plotted on the y-axis and streptavidin signal is plotted on the x-axis. The plots were gated on viable singlet cells, and the fluorescence thresholds were determined using the negative controls (unstained cells and stained cells labeled with unbiotinylated Avitagged CD4 YW -Ig). The proportion of double-positive cells for CD19 and CD4-Ig are indicated in the top right quadrant. h , Graphic of the tonsil organoid culture experimental set-up. Five to six million tonsil cells were aliquoted in 12-well transwell plates with each well containing a 1 mL outer reservoir of media. 20 μL from the outer reservoirs were sampled at 2 hours, 24 hours, 48 hours, 72 hours, and 96 hours post-transwell seeding. The outer reservoirs were mixed by pipetting prior to each collection and the timepoints were stored at -80 °C. i, j, Time-course measurements (left) of the concentration of CD4 YW -Ig and gCD4 YW -Ig incubated in tonsil culture media without tonsil cell seeding ( i ) or with tonsil cell seeding ( j ) and their area under the curves (right). Concentrations were determined by ELISA in technical triplicates using the respective proteins as standards. Each point is an average of three independent transwell samples and the error bars are s.d. Variants containing the Q40Y and T45W substitutions are denoted in subscript. CD4 YW -Ig for i and j additionally contain the thermostabilizing A55V, L109K, L151K, and L177E substitutions. gCD4 constructs for e, g, i , and j contain A55V, S60D, D63K, L109K, L151K, and L177E substitutions. Donor #664 was used for the data in b-g and donor #669 was used for the data in i and j .

Article Snippet: Samples were pelleted and resuspended in 50 μL of FACS buffer containing streptavidin-Alexa Fluor 488 conjugate (S32354; Invitrogen) diluted 1:50 (final concentration 40 μg/mL).

Techniques: Flow Cytometry, Isolation, Staining, Blocking Assay, Labeling, Fluorescence, Concentration Assay, Incubation, Enzyme-linked Immunosorbent Assay, Construct

Journal: Nature Communications

Article Title: Tumor factors stimulate lysosomal degradation of tumor antigens and undermine their cross-presentation in lung cancer

doi: 10.1038/s41467-022-34428-w

Figure Lengend Snippet: a Merged MFI expression of SIINFEKL-bound H-2kb in DCs from indicated mice after treatment of OVA protein (200 μg/mL, 18 h). n = 3 biologically independent samples. b Percentage of IFNγ + in OT-I CD8 + T cells after 72 h co-culture of naïve OT-I CD8 + T cells with splenic CD11c + myeloid cells from indicated mouse pulsed with OVA (10:1) or MC38-OVA lysate (10:4). n = 3 biologically independent samples. c Antigen cross presentation analysis for WT or Ch25h −/− DCs pre-treated or not with 25HC (50 nM, 4 h before adding soluble sOVA at indicated concentrations). OVA-pulsed DCs were then co-cultured (10:1 for 72 h) with OT-I CD8 + T cells labeled with carboxy fluorescein succinimidyl ester (CFSE). Proliferation of these T cells was assessed by CFSE dilution. n = 3 biologically independent samples. d Antigen cross presentation analysis for WT or Ch25h −/− DCs pre-treated or not with 25HC (50 nM, 4 h before adding beads loaded with OVA at indicated percentage) was carried out as in panel C. n = 3 biologically independent samples. e Antigen cross presentation analysis for Ch25h −/− DCs transduced with retroviruses for expression of CH25H WT or catalytically inactive CH25H H242,243Q mutant. n = 4 biologically independent samples. f Antigen cross presentation analysis for WT DCs pre-treated with or without PGE 2 (10 ng/mL), VEGF (20 ng/mL) or TCM from LLC cells (75%, v/v) for 24 h with or without the treatment of 25HC (50 nM) or DC661 (5 μM) for 4 h as indicated. Then DCs were pulsed with soluble OVA protein (50 or 100 μg/mL, 6 h) and co-cultured with CFSE-labeled OT-I T cells (10:1 for 72 h). n = 3 biologically independent samples. g Antigen cross presentation analysis for WT DCs pre-treated with or without PGE 2 (10 ng/mL), VEGF (20 ng/mL) or TCM from LLC cells (75%, v/v) for 24 h with or without the treatment of 25HC (50 nM) or DC661 (5 μM) for 4 h as indicated. Then DCs were then treated with beads-bound OVA protein (25% or 50%, 1 h) and co-cultured with CFSE-labeled OT-I T cells (10:1 for 72 h). n = 3 biologically independent samples. h Antigen cross presentation analysis for DCs of indicated genotypes. T cell proliferation was assessed by flow cytometry in CFSE-labeled OT-I T cells co-cultured (10:1 for 72 h) with DCs from indicated mice, pretreated with conditioned media from LLC cells (70%, v/v) for 24 h and pulsed with soluble OVA protein (200 μg/mL, 6 h). n = 5 biologically independent samples. h Data are presented as mean ± SEM. Statistical analysis was performed using 2-tailed Students’ t -test (A, B, C, D, E and H) or 1-way ANOVA with Tukey’s multiple-comparison test (F and G) test. n.s., not significant. Source data are provided as a Source Data file.

Article Snippet: Cells were washed and resuspended in staining buffer or PBS and either analyzed on LSRFortessa flow cytometry (BD Biosciences) or Canto flow cytometry (BD Bioscience).

Techniques: Expressing, Co-Culture Assay, Cell Culture, Labeling, Transduction, Mutagenesis, Flow Cytometry

Journal: Nature Communications

Article Title: Tumor factors stimulate lysosomal degradation of tumor antigens and undermine their cross-presentation in lung cancer

doi: 10.1038/s41467-022-34428-w

Figure Lengend Snippet: a Kaplan–Meier analysis of survival of LLC tumor-bearing mice after intravenous injection of 4 × 10 5 LLC by log-rank test. n = 8 mice in Ch25h f/f and n = 9 mice in Ch25h ΔDC groups. b Tumor weight, representative lung images and the corresponding H&E-stained lung sections from Ch25h f/f and Ch25h ΔDC mice ( n = 4 mice per group) 18 days after intravenous injection of 6 × 10 5 LLC tumor cells. Scale bar:2 mm. Similar results were obtained from three independent experiments. c Flow-cytometric determination of the percentage and quantitative estimates of intratumoral CD8 + T cells in tumor lungs from Ch25h f/f and Ch25h ΔDC mice. n = 6 tumors per genotype. d Tumor weight, representative lung images and the corresponding H&E-stained lung sections from mice of indicated genotypes ( n = 5 mice per group) 17 days after intravenous injection of 1 × 10 6 LLC tumor cells. Scale bar: 1 mm. Similar results were obtained from three independent experiments. e Volume, appearance and mass of LLC tumors on Day 17 after s.c. inoculation (5 × 10 5 cells/mouse) into mice of indicated genotypes. n = 5 mice per group. f Flow cytometry analysis of IFN-γ expression by CD8 + T cells isolated from LLC tumors or spleens of LLC-bearing mice from experiments described in Panel 6e. g Growth of LLC tumors in mice after s.c. injection of 6 × 10 5 LLC cells into Ch25h f/f and Ch25h ΔDC mice. After 8 days of LLC inoculation, animals were treated with PBS or gemcitabine (GEM, 30 mg/kg, every 3 days, four times) plus cisplatin (CDDP, 3 mg/kg, every 6 days, twice). Tumor weight was measured at day 20 from tumor inoculation. n = 5 mice per group, data was shown in Mean ± SEM. h Flow-cytometric analysis of the percentage and quantitative estimates of intratumoral CD8 + T, Ki67 + and GzmB + CD8 + T cells. n = 5 tumors in each group. i Volumes of LLC tumors in syngeneic mice that were either vaccinated with LLC cells undergoing immunogenetic cell death (ICD) or not vaccinated. Data were processed using the mixed-effect analysis of 2-way ANOVA with Tukey’s multiple comparisons test. n = 5 mice per group. j Kaplan–Meier analysis of survival of mice from experiment described in panel i . Data were processed using the Log-rank test. n = 5 mice per group. Data presented as mean ± SEM. Statistical analysis was performed using 1-way ANOVA with Tukey’s.l multiple-comparison test (D, E, F, G and H) test or 2-way ANOVA with multiple comparison (E, G and I) or 2-tailed Students’ t -test (B and C) or Kaplan-Meier survival analysis (A and J). n.s., not significant. Source data are provided as a Source Data file.

Article Snippet: Cells were washed and resuspended in staining buffer or PBS and either analyzed on LSRFortessa flow cytometry (BD Biosciences) or Canto flow cytometry (BD Bioscience).

Techniques: Injection, Staining, Flow Cytometry, Expressing, Isolation

Journal: Nature Communications

Article Title: Tumor factors stimulate lysosomal degradation of tumor antigens and undermine their cross-presentation in lung cancer

doi: 10.1038/s41467-022-34428-w

Figure Lengend Snippet: a qPCR analysis of Ch25h mRNA in BMDCs pre-treated with MSA-2 (10 µM) for 30 min and LLC-conditioned media (TCM, 67%, v/v ) for 2 h. n = 3 biologically independent samples. b Quantitative estimates of lysosome activity in WT or Ch25h −/− BMDCs pretreated with LLC-conditioned media (TCM, 70% v/v) with or without pre-treatment of MSA-2 (10 µM, 2 h pretreatment) for 16 h. n = 4 biologically independent samples. c Antigen cross-presentation was assessed by proliferation of CFSE-labeled OT-I T cells co-cultured (10:1) with OVA-pulsed (200 μg/mL, 6 h) WT or Ch25h −/− DCs for 72 h. DCs were pre-treated with medium conditioned by LLC cells (70%, v/v) for 20 h with or without the treatment of MSA-2 (10 µM) for 16 h before OVA pulse. n = 4 mice per group. d Volumes of LLC tumors grown in Ch25h f/f (top panel) or Ch25h ∆DC (bottom panel) mice treated with Vehicle+Isotype control (anti-mouse IgG1 monoclonal antibody), anti-PD1 antibody (i.p, 5 mg/kg every 4 days), MSA-2 (orally, 60 mg/kg every 4 days) and combination. n = 5 mice per group. e Tumor mass of LLC tumors from experiment described in panel d . n = 5 mice per group. f Kaplan–Meier analysis of survival of LLC tumor-bearing mice described in panel d . n = 5 mice per group. g Flow cytometry analysis of number of CD3 + CD8 + T cells in LLC tumors grown in Ch25h f/f or Ch25h ∆DC mice with indicated treatment described in panel d . n = 5 mice per group. h Flow cytometry analysis of the percentage of CD8 + PD-1 + , CD8 + LAG3 + and CD8 + TIM3 + T cells in tumors from experiment described in panel d . n = 5 mice per group. Data presented as mean ± SEM. Statistical analysis was performed using 2-tailed Students’ t -test (A, B, C, E, G and H) or 2-way ANOVA with multiple comparison (D) or Kaplan-Meier survival analysis (F). n.s., not significant. Source data are provided as a Source Data file.

Article Snippet: Cells were washed and resuspended in staining buffer or PBS and either analyzed on LSRFortessa flow cytometry (BD Biosciences) or Canto flow cytometry (BD Bioscience).

Techniques: Activity Assay, Labeling, Cell Culture, Flow Cytometry