Journal: Cancers

Article Title: Pancreatic Cancer Cells Undergo Immunogenic Cell Death upon Exposure to Gas Plasma-Oxidized Ringers Lactate

doi: 10.3390/cancers15010319

Figure Lengend Snippet: Gas plasma-oxidized Ringer’s lactate induces immunogenic cell death in pancreatic cancer cells. ( a ) A schematic overview of the in vitro experimental procedure with ROS production (purple), cell death (blue), and receptor expression analyses (schematic bubbles on the right) to indicate modes of analysis in this study; ( b , c ) percentage of MitoSOX Red positive ( b ) KPC960-GFP/Luc and ( c ) PDA6606 cells after exposure to serial dilutions of oxRilac solutions; ( d – f ) representative flow cytometry density plots ( d ) of cellular viability after oxRilac exposure and quantification thereof for ( e ) KPC960-GFP/Luc and ( f ) PDA6606 cells (IC 25 (dashed) and IC 50 (continuous) were indicated as red lines for each cell line); ( g ) representative flow cytometry intensity histograms of calreticulin (CRT), heat shock protein (HSP) 70, and HSP90 expression of KPC960-GFP/Luc cells; ( h ) percentage of positive cells; ( i ) representative flow cytometry intensity histograms of (CRT), HSP70 and HSP90 expression of PDA6606 cells; ( j ) percentage of positive cells. Data are mean ± standard error (SEM). Heat maps show medians. Statistical analyses were performed using two-way analyses of variance (* p <0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: For the analysis of immunogenic surface markers, cells were stained with antibodies (conjugate) targeted against calreticulin (APC-Cy7; Novus Biologicals, Wiesbaden, Germany), heat shock protein (HSP) 70 (PE/Texas Red; Santa Cruz, Heidelberg, Germany), and HSP90 (APC; Thermo Fisher Scientific, Dreieich, Germany) for 30 min at 37 °C.

Techniques: Clinical Proteomics, In Vitro, Expressing, Flow Cytometry

Journal: Cells

Article Title: Chronic Pain Induced by Social Defeat Stress in Juvenile Mice Depends on TLR4

doi: 10.3390/cells14050350

Figure Lengend Snippet: SDS increases TLR4 dimers of spinal cord microglia and DRG macrophages. ( A ) TLR4 dimers (%) in SDS and no-SDS groups of microglia (CD11b + population) and ( B ) macrophages (CD11b + /F4-80 + population) on day 22 of the protocol. ( C ) Gating strategy for lumbar spinal cord cells, first live cells were gated (APC-Cy7 live/dead Ghost dye); second, microglia population (PercpCy 5.5-conjugated CD11b+), lastly—TLR4 monomers (PC7-conjugated TLR4/MD2) or TLR4 total (APC-conjugated total TLR4). ( D ) Gating strategy for dorsal root ganglia cells, first live cells were gated (APC-Cy7 live/dead Ghost dye); second, microglia population (PercpCy 5.5-conjugated CD11b+), third macrophages population (488-conjugated F4-80) and lastly—TLR4 monomers (PC7-conjugated TLR4/MD2) or TLR4 total (APC-conjugated total TLR4). Data represent mean ± SEM, t -test, SDS vs. no-SDS, * p < 0.05, ** p < 0.01, n = 6 (SDS), and n = 6 (no-SDS).

Article Snippet: Single-cell suspensions were incubated for 45 min on ice with the following antibodies: APC-Cy7 live/dead Ghost dye (#18452, Cell Signaling, Danvers, MA, USA) or 540 live/dead Ghost dye (#72086, Cell Signaling, Danvers, MA, USA); PC7-conjugated TLR4/MD2 (#MTS510; ThermoFisher, Waltham, MA, USA); APC-conjugated total TLR4 (#SA15-21; Biolegend, San Diego, CA, USA); 488-conjugated F4-80 (#123110; Biolegend, San Diego, CA, USA); and PercpCy 5.5-conjugated CD11b (#101228; Biolegend, San Diego, CA, USA).

Techniques:

Journal: Nutrients

Article Title: Annona muricata L.-Derived Polysaccharides as a Potential Adjuvant to a Dendritic Cell-Based Vaccine in a Thymoma-Bearing Model

doi: 10.3390/nu12061602

Figure Lengend Snippet: Immunization with ALP-treated DCs induces a robust anti-tumor effect and immunity. ( A ) Two weeks after the final immunization, single cells obtained from PBS-, iDC-, OVAs-DC-, and ALP/OVAs-DC-immunized mice were stimulated ex vivo with OVA peptides for 6 h. The percentage of OVA-specific CD3 + CD4 + and CD3 + CD8 + T cells producing CD107a, IFN-γ, IL-2, or TNF-α was analyzed in accord with the gating strategy described in . Pie charts indicate the mean frequencies of CD4 + and CD8 + T cells co-expressing IFN-γ, TNF-α, IL-2, and CD107a. ( B ) Serum samples were analyzed using ELISA for OVA 323–339 -specific IgG2a and IgG1 antibodies in each immunized mouse. The mean ± SD ( n = 6 mice/group) shown are representative of 2 independent experiments. ( C ) Tumor volumes in mice ( n = 10 mice/group) that received PBS (G1: white solid dot), iDC (G2: green solid dot), OVAs-DC (G3: red solid dot), and ALP/OVA-DCs (G4: blue solid dot) vs. E.G7. Tumor growth was identified by calculating the diameter of the tumor every 3 days for 30 days. The mean ± SD shown are representative of 2 independent experiments. SD; standard deviation.

Article Snippet: The cells were then stained using Live/Dead and with anti-CD3 (APC-Cy7, eBioscience), anti-CD4 (Alexa488), and anti-CD8 (PerCp-Cy5.5, eBioscience) mouse Abs (mAbs) for 30 min at 4 °C.

Techniques: Ex Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation