Journal: Clinical Epigenetics

Article Title: DNA methylation regulates TIGIT expression within the melanoma microenvironment, is prognostic for overall survival, and predicts progression-free survival in patients treated with anti-PD-1 immunotherapy

doi: 10.1186/s13148-022-01270-2

Figure Lengend Snippet: Flow cytometry analysis of TIGIT expression of pharmacologically demethylated melanoma cells. Representative SSC/FSC and FSC/TIGIT-PerCP/Cyanine5.5 dot plots of untreated and unstained ( a ), untreated ( b ), and 5‐aza‐dC treated A375 melanoma cells ( c ). d Flow cytometric histograms showing TIGIT expression of pharmacologically demethylated (5‐aza‐dC treated) compared to untreated A375 melanoma cell line

Article Snippet: A375 melanoma cell line pellets were washed with flow cytometry buffer (1X Dulbecco’s Phosphate Buffered Saline [cat. no. 14190094, Thermo Fisher Scientific], 4% [v/v] FBS, 2 mM ethylenediaminetetraacetic acid [EDTA]).

Techniques: Flow Cytometry, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Function and Role of Intercellular Adhesion Molecule 2 in Dental Pulp Cells and Tissue

doi: 10.3390/ijms262412006

Figure Lengend Snippet: ICAM2 localization in rat dental pulp tissue and ICAM2 expression in HDPCs. ( A ) The mRNA expression of ICAM1 , ICAM2 (black column), ICAM3 , ICAM4 , ICAM5 in HDPC-5Y was assessed by quantitative RT-PCR (qRT-PCR). It was normalized against β-actin expression (means ± SD; n = 4; ** p < 0.01). ( B ) Hematoxylin-eosin (H&E) staining of tissue sections (sagittal sections) of mandibular first molars from Wistar rats. The right panel is the higher magnification view of boxed area in the left panel. PU: dental pulp tissue; De: Dentin. Bars, 100 μm. ( C – E ) Immunofluorescence (IF) staining of ICAM2 in the normal dental pulp tissue ( C ). The higher magnification view of boxed area in ( C , D ). Positive staining was indicated by arrow heads (odontoblasts) and arrow (dental pulp cells). Negative control: rabbit IgG (cIgG; ( E )). Nuclei were stained with DAPI (Blue). Bars, 100 μm. ( F , G ) The expression of ICAM2 in HDPC-5Y was examined by IF staining. Anti-ICAM2: Green ( F ), anti-rabbit IgG (control IgG: cIgG; ( G )). Nuclei were stained with DAPI (Blue). Bars, 100 μm. Arrow heads indicate ICAM2-positive HDPCs ( F ). ( H ) The gene expression of ICAM2 in three HDPCs (HDPC-5Y, 5L, and 5I) was examined by semi-qRT-PCR. It was normalized against GAPDH expression. ( I ) The expression intensities of ICAM2 (CD102) of HDPC-5Y (orange line) were demonstrated by flow cytometry. In the gated region, positive cells. Gray line indicates negative control (rabbit IgG).

Article Snippet: Cells (2 × 10 5 /tube) were prepared as a single cell suspension by trypsin/EDTA digestion and resuspended in flow cytometry buffer (R&D Systems, Minneapolis, MN, USA), then incubated with antibodies (10 mg/mL) specific for surface markers or isotype control antibodies (10 mg/mL) on ice for 45 min. Anti-CD102 (ICAM2)-PE antibodies (eBioscience, San Diego, CA, USA) and mouse IgG-PE isotype control were used.

Techniques: Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, Negative Control, Control, Gene Expression, Flow Cytometry

Journal: Biomaterials

Article Title: Robust tolerogenic dendritic cells via push/pull pairing of toll-like-receptor agonists and immunomodulators reduces EAE

doi: 10.1016/j.biomaterials.2022.121571

Figure Lengend Snippet: LipoTLR + I generate tolDCs that actively uptake antigen in vitro and in vivo. (A) Analysis of LipoTLR + I In Vitro. 100 k BMDCs were treated with free or liposomal formulations of TLR + I, TLR or I and analyzed via flow cytometry for liposomal uptake. Liposomes were synthesized with AF647-OVA for a total of 1 μg of OVA per .1 μM of inhibitor (using loading procedure from Figure S-4). Free inhibitor formulations were treated with equivalent OVA dose. 1 h after second treatment, cells were washed and analyzed via flow for OVA internalization (B) In Vivo Analysis of LipoTLR + I uptake. C57BL/6 mice (4 per group) were injected with either free or Lipo formulations of OVA and combinations of inhibitors to for the following categories (100 μg OVA/mouse, 10 umols inhibitor/mouse, 1 μg FLA/mouse, 10 μg CpG/mouse) [1]: OVA alone (PBS) [2], OVA + TLR agonists (TLR), OVA + TLR agonists + dexsamethasone only (TLR + Dex) or OVA + Inhibitor combination (TLR + I). For Lipo formulations, DiD was added at 0.01% total lipid loading to allow fluorescent analysis. Mice were injected with either Lipo or free combinations i.p. with FLA then CpG formulations on consecutive days. 24 h after final CpG injection, mice were sacrificed, popliteal and inguinal lymph nodes harvested, disassociated and stained for various immune cell markers. Lymph cells were analyzed via spectral flow and DC populations (CD45+, MHCII+, CD11c+, CD19−) analyzed for CD40, (C) CD80 (D) CD86 (E) CD103 (F) PD-L1 (G) PD-L2. (H) Mice treated with liposomes were gated on Liposome+ and PD-L1/2 + cell populations. Error bars indicate ± of SD of each mouse group (N = 4). Significance was determined by a two-way ANOVA with Tukey post hoc test for multiple comparisons. *p < 0.5, **p < 0.01, ***p < 1 × 10−4, ****p < 1 × 10−5..

Article Snippet: Splenocytes were then washed using flow cytometry buffer (Stem Cell Technologies), stained for live/dead (Live Dead Aqua) for 30 min, washed and stained for various cell surface markers for 1 h. Cells were washed and analyzed using an Aurora Spectral Flow Cytometer. .

Techniques: In Vitro, In Vivo, Flow Cytometry, Liposomes, Synthesized, Injection, Staining

Journal: The Journal of Experimental Medicine

Article Title: Human lymphoma mutations reveal CARD11 as the switch between self-antigen–induced B cell death or proliferation and autoantibody production

doi: 10.1084/jem.20112744

Figure Lengend Snippet: A potent CARD11 mutation blocks self-antigen–induced B cell death and promotes extensive proliferation in vivo. (a) Experimental strategy to examine the consequences of acquiring different CARD11 coiled-coil domain mutations in normal antigen-activated B cells. (b) Immunoblot for phospho/total p65 and phospho/total JNK in cell lysates from EV, WT, Mut10, and constitutively active IKK-β (IKK-β*)–expressing B cells. Cells cultured in media without anti-CD40 for 24 h before EGFP + cells were sorted. (c) Antigen-specific B cells transduced with the indicated vectors were transplanted into Rag1 -deficient recipient mice that were either nontransgenic, in which the B cells lacked antigen or CD40 stimuli, or were HEL transgenic, in which the transplanted B cells were continuously stimulated by circulating self-antigen. Donor cells in the spleen of recipient mice were analyzed by flow cytometry 5 and 12 d after transplantation. In the top panels, the transplanted B cells were detected and enumerated as a percentage of spleen cells by their binding of HEL antigen and staining for IgM a allotype. The bottom panels are gated on these transplanted B cells and show the percentage that are EGFP + and their expression of CD19. (d) The top panel shows the number of EGFP + B cells that were transplanted into each mouse (input). Bottom panels show mean number ± SEM of EGFP + B cells in the spleen of nontransgenic or HEL transgenic recipient mice on days 5 and 12 ( n = 3–6 recipient mice per group and time point; one way analysis of variance [ANOVA]: P = 0.0011 and P = 0.0014, respectively). Data are representative of four independent experiments.

Article Snippet: Samples were washed twice, resuspended in flow cytometry buffer, and analyzed with an LSR II or LSR Fortessa (BD).

Techniques: Mutagenesis, In Vivo, Western Blot, Expressing, Cell Culture, Transduction, Transgenic Assay, Flow Cytometry, Transplantation Assay, Binding Assay, Staining

Journal: The Journal of Experimental Medicine

Article Title: Human lymphoma mutations reveal CARD11 as the switch between self-antigen–induced B cell death or proliferation and autoantibody production

doi: 10.1084/jem.20112744

Figure Lengend Snippet: A range of lymphoma CARD11 mutations switch self-antigen–induced B cell deletion into aberrant B cell growth. (a) Immunoblot and densitometry analysis of CARD11 in cell lysates from cells expressing control EV and WT CARD11 and lymphoma CARD11 mutants Mut2, Mut3, Mut6, and Mut10. Transduced B cells were cultured in media without anti-CD40 for 24 h before EGFP + cells were sorted. (b) BIM levels expressed as mean fluorescence intensity (MFI) measured by flow cytometry analysis of transduced B cells with the indicated vectors (one-way ANOVA: P < 0.0001). (c) Number of input EGFP + Ig transgenic B cells, transduced with vectors encoding the indicated CARD11 alleles, that were transplanted into each recipient mouse in panels d–f. (d) Flow cytometric analysis of splenocytes 12 d after transplantation of B cells, transduced with vectors encoding the indicated CARD11 alleles, into HEL transgenic or nontransgenic recipients. B220 expression and the percentage of EGFP + B cells, either as a percentage of all splenocytes or (in parentheses) as a percentage of the transplanted B cells, are shown. (e) Mean number (±SEM; n = 3–6 mice per group) of splenic EGFP + HEL-binding IgM a+ B cells in HEL transgenic or nontransgenic recipients 12 d after transplantation of transduced B cells (one-way ANOVA: P < 0.0001). (f) Relative units of anti-HEL IgM measured by ELISA in serum collected from nontransgenic or HEL transgenic mice 11 d after transplantation ( n = 3–9 mice per group; one-way ANOVA: P < 0.0001). (g) Mean number (±SEM; n = 3–5 mice per group) of splenic EGFP + HEL-binding IgM a+ B cells in HEL transgenic or nontransgenic recipients, 11 d after transplantation of Ig transgenic or anergic Ig × HEL double transgenic B cells (one-way ANOVA: P < 0.0001). (h) Relative units of anti-HEL IgM measured by ELISA on serum collected from nontransgenic or HEL transgenic mice 10 d after transplantation ( n = 3–5 mice per group; one-way ANOVA: P < 0.0001). Data are representative of three independent experiments.

Article Snippet: Samples were washed twice, resuspended in flow cytometry buffer, and analyzed with an LSR II or LSR Fortessa (BD).

Techniques: Western Blot, Expressing, Cell Culture, Fluorescence, Flow Cytometry, Transgenic Assay, Transduction, Transplantation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay