Journal: Cancers

Article Title: CAR-NK Cells Targeting HER1 (EGFR) Show Efficient Anti-Tumor Activity against Head and Neck Squamous Cell Carcinoma (HNSCC)

doi: 10.3390/cancers15123169

Figure Lengend Snippet: Characterization of primary HNSCC (pHNSCC) cells. Characterization of ( A ) CD45 + immune cells present in isolated pHNSCC samples as well as ( B ) percentage of NK cells (CD56 + ), macrophages (CD11b + ), B cells (CD19 + ) and T cells (CD3 + ) of CD45 + cells in pHNSCC samples and ( C ) percentages of stroma cells (CD140a + ), endothelial cells (CD31 + ), cancer-associated fibroblasts (FAP + ) and epithelial marker-positive cells (EpCAM + ) in the CD45 − pHNSCC cell population within 24 h from isolation (or thawing). ( D ) Target antigen expression within 24 h of isolation (or thawing) of pHNSCC samples. ( E ) Quantitative analysis of immunohistochemical stainings of seven patient samples (photos from exemplary patient samples shown in ). ( F ) HER1 expression on EpCAM + sorted and expanded pHNSCC samples. Optical genome mapping of EpCAM + sorted and expanded pHNSCC samples ( G ) #14 and ( H ) #16. Red box highlights amplification of 11q13, (Legend: ● copy number gains (blue lines), ● copy number losses (red lines), ● insertions, ● deletions, ● inversions, ● duplications, ● intra-fusions or inter-translocations (pink lines)). Mean with standard deviation shown. n = 4–9 pHNSCC samples as indicated ( A – E ).

Article Snippet: Next, cells were incubated with anti-Flag PE (BioLegend, San Diego, CA, USA) and anti-CD56 PC7 (BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Isolation, Marker, Expressing, Immunohistochemical staining, Amplification, Standard Deviation

Journal: Scientific Reports

Article Title: Immediate systemic neuroimmune responses following spinal mobilisation and manipulation in people with non-specific neck pain: a randomised placebo-controlled trial

doi: 10.1038/s41598-023-39839-3

Figure Lengend Snippet: Neuroimmune parameters. ( A ) Measured using multianalyte assay Ella (R&D systems, Minneapolis, United States) Cardiac C-Reactive Protein (Latex) High Sensitive using R oche/Hitachi cobas c systems. Markers TNF-α (Inter-assay coefficient of variation: 4.27%), sTNF-R2 (5.78%), IL-1β (4.97), IL-1RA (7.20%) directly from blood samples measured using multianalyte assay Ella (R&D systems, Minneapolis, United States). Aliquots of blood samples to determine ex-vivo levels of inflammatory markers were stored at − 80 °C after centrifugation for 10 min at 1530 g . ( B ) Stimulated for 24 h at 37 °C, in a humidified 5% CO 2 incubator, with lipopolysaccharide (LPS) from Escherichia coli O55:B5 at a concentration of 1 ng/ml and 10 µg/ml. Determined using a custom-made U-plex (MSD, Maryland, United States) Whole blood was stimulated with high dose (10 µg/ml (HD-LPS)) or low dose (1 ng/ml) LPS. Supernatant was diluted 100-fold and tested for TNF-α (Inter-assay coefficient of variation: TNF-α (7%), IL-1β (12.7%), IL-1RA (10.6%), IL-10 (22%), CCL2, (8.4%), CCL3 (12.7%), CCL4 (12.9%) using the above-mentioned U-plex. ( C ) Determined by 10-color flowcytometry (FCM): CD45+ = General Leukocyte marker; CD3+ = T-cell marker; CD3+CD4+ = CD4+ T-helper marker; CD3+CD4+CD25hi = T-regulator cell marker; CD3+CD8+ = Cytotoxic T-cell marker; CD3-CD56+ = Natural Killer cell marker; CD19+ = B-cell marker; CD14+ = monocyte marker; HLA-DR = activation marker for T-cells and monocytes; TLR-4 = Toll-like receptor 4 marker. CD25+ = activation marker for T-cells, Fluorescence-activated cell sorting (FACS) staining was used for cell surface staining of mononuclear cells using a whole blood staining protocol and red blood cell lysis using optilyse B conform manufacturer recommendation (Beckman Coulter, Brea, CA). For quantification of lymphocyte subsets Trucount tubes were used (BD Biosciences, Franklin Lakes, NJ). The following monoclonal antibodies were used: CD8-APC-AF700 (B9.11), CD19-ECD (J3-119), CD56-PC7 (N901) all from Beckman Coulter; HLA-DR-FITC (G46-6), CD14-APC (M5E2), TLR4-PE (TF901) all from BD Pharmingen (San Diego, CA) and CD3-APC (SK7), CD4-APC-H7 (SK3), CD25-PE (2A3), CD45-PerCP (2D1) all from BD Biosciences. HLA-DR was used as activation marker for T-cells and monocytes, CD25 was used as activation marker for T-cells; TLR-4 expression was assessed on monocytes. Isotypes were used as control for these activation markers. Samples were run on FACS Gallios (Beckman Coulter) and analyzed using Kaluza (Beckman Coulter). The total number of leucocyte was determined using Z2 analyzer (Beckman Coulter). TNF-α: Tumor Necrosis Factor-α; TNF-RII: Tumor Necrosis Factor Receptor Antagonist 2; IL-1β: Interleukin-1β; IL-1RA: Interleukin-1 receptor antagonist; hsCRP: High sensitive C-Reactive Protein; IL-4: Interleukin-4; IL-10: Interleukin-10; CCL2: c–c-motif chemokine ligand 2; CCL3: c–c-motif chemokine ligand 3; CCL4: c–c-motif chemokine ligand 4; CD: cluster of differentiation.

Article Snippet: The following monoclonal antibodies were used: CD8-APC-AF700 (B9.11), CD19-ECD (J3-119), CD56-PC7 (N901) all from Beckman Coulter; HLA-DR-FITC (G46-6), CD14-APC (M5E2), TLR4-PE (TF901) all from BD Pharmingen (San Diego, CA) and CD3-APC (SK7), CD4-APC-H7 (SK3), CD25-PE (2A3), CD45-PerCP (2D1) all from BD Biosciences.

Techniques: Inter Assay, Ex Vivo, Centrifugation, Concentration Assay, Marker, Activation Assay, Fluorescence, FACS, Staining, Lysis, Expressing