Journal: International Journal of Molecular Sciences

Article Title: Neutrophils in the Spotlight—An Analysis of Neutrophil Function and Phenotype in ARDS

doi: 10.3390/ijms252312547

Figure Lengend Snippet: Overview of fluorescently labeled antibodies. Abbreviations: phycoerythrin (PE), fluorescein isothiocyanate (FITC), allophycocyanin (APC).

Article Snippet: fMLP-Receptor , APC , APC Anti-human, fMLP receptor Antibody Clone: REA169 Miltenyl Biotec, Bergisch Gladbach, Deutschland.

Techniques: Labeling

Journal: International Journal of Molecular Sciences

Article Title: Neutrophils in the Spotlight—An Analysis of Neutrophil Function and Phenotype in ARDS

doi: 10.3390/ijms252312547

Figure Lengend Snippet: Overview of fluorescently labeled antibodies. Abbreviations: phycoerythrin (PE), fluorescein isothiocyanate (FITC), allophycocyanin (APC).

Article Snippet: fMLP-Receptor , APC , APC Anti-human, fMLP receptor Antibody Clone: REA169 Miltenyl Biotec, Bergisch Gladbach, Deutschland.

Techniques: Labeling

Journal: International Journal of Molecular Sciences

Article Title: Neutrophils in the Spotlight—An Analysis of Neutrophil Function and Phenotype in ARDS

doi: 10.3390/ijms252312547

Figure Lengend Snippet: Overview of fluorescently labeled antibodies. Abbreviations: phycoerythrin (PE), fluorescein isothiocyanate (FITC), allophycocyanin (APC).

Article Snippet: fMLP-Receptor , APC , APC Anti-human, fMLP receptor Antibody Clone: REA169 Miltenyl Biotec, Bergisch Gladbach, Deutschland.

Techniques: Labeling

Journal: Cancer Discovery

Article Title: A TLR3 Ligand Reestablishes Chemotherapeutic Responses in the Context of FPR1 Deficiency

doi: 10.1158/2159-8290.cd-20-0465

Figure Lengend Snippet: Figure 1. Screening for genes that modulate antigen cross-presentation. A, Cas9-expressing iniDCs (iniDC_Cas9) were transfected with specific gRNA to perform gene knockouts. Dex/Dox were removed from the culture system during transfection and recovery of cells to differentiate iniDC_Cas9 cells into primary de-iniDC_Cas9 DCs, which were then pulsed with OVA and cocultured with B3Z hybridoma cells. IL2 secretion was measured as an indica- tor for antigen cross-presentation capacity. B, Ranked dot plots (mean ± SEM, n = 2) depict the IL2 concentration. Non–antigen-loaded and SIINFEKL peptide–loaded iniDCs were used as negative and positive controls for the in vitro cross-presentation assay. Nontargeted gRNA was used as transfec- tion control. C–E, Immunocompetent C57BL/6 mice were inoculated s.c. with WT murine MCA205 cells. When tumor became palpable, mice received i.t. injection of antigen-pulsed de-iniDC_Cas9 DCs (PBS, as WT control) or Fpr1−/− de-iniDC_Cas9 DCs (Fpr1−/− DCs; C) every 4 days. In addition, for antibody- mediated depletion of ANXA1 (D), de-iniDCs were resuspended in solutions containing 250 μg/mL of isotype control (PBS or WT DCs) or anti-ANXA1 (αANXA1) antibodies (αAnxA1 and αAnxA1 DCs). For the depletion of T cells (E), 200 μg of isotype control (PBS or WT DCs), or 100 μg of monoclonal anti-CD4 (αCD4) plus anti-CD8 (αCD8) antibodies (αCD4+αCD8 and αCD4+αCD8 DCs) were injected i.p. on the same day, 1 day, 1 week, and 2 weeks after de-iniDC administrations. Results from one experiment involving 6 mice/group. Data are represented as mean ± SEM over time (C–E). *, P < 0.05; **, P < 0.01; ****, P < 0.0001 as compared with PBS. ###, P < 0.001; ####, P < 0.0001 as compared with WT DCs.

Article Snippet: WT and Fpr1−/− mice were sacrificed, and tumors were harvested and placed in ice-cold RPMI (1 mL) in GentleMACS C tubes (Miltenyi Biotec).

Techniques: Expressing, Transfection, Concentration Assay, In Vitro, Control, Injection

Journal: Cancer Discovery

Article Title: A TLR3 Ligand Reestablishes Chemotherapeutic Responses in the Context of FPR1 Deficiency

doi: 10.1158/2159-8290.cd-20-0465

Figure Lengend Snippet: Figure 2. Compensation of Fpr1 deficiency in DCs with NOD or TLR agonists. A, WT and Fpr1−/− iniDCs were differentiated into de-iniDCs with the characteristics of primary DCs, which were then incubated with soluble OVA, in the presence of the indicated NOD or TLR agonists, at different concentrations (briefly, 2-fold serial dilutions of maximum 4 μg/mL for C123-IE-DAP, MDP, LPS-B5, pIC, Pam3CSK4, and R848; 2 μg/mL for FLA-BS and 2 μmol/L for ODN1826) for 6 hours, and cocultured with B3Z cells for another 18 hours in the presence of the same treatments following the washing step. B, The coculture supernatant was collected for ELISA quantification of IL2 secretion. IL2 concentration was normalized both between the lowest and highest values from the standard and between the untreated Fpr1−/− de-iniDCs and WT de-iniDCs. Data are represented as bar charts (mean ± SD, n = 3). *, P < 0.05; **, P < 0.01 (multiple Welch t test with Bonferroni correction) as compared with WT-untreated de-iniDCs. #, P < 0.05; ##, P < 0.01; ###, P < 0.0001 (multiple Welch t test with Bonferroni correction) as compared with Fpr1−/−-untreated de-iniDCs. C, Cas9-expressing iniDCs were transiently transfected with specific gRNA to Fpr1 alone, Tlr3 alone, or their combinations for single/double gene knockouts. A nontarget control gRNA was used as an internal control. Dex/Dox were removed from the culture system during transfection and recovery of cells to differentiate iniDC_Cas9 cells into primary de-iniDC_Cas9 DCs. De-iniDC_Cas9 DCs were treated with 0.5 μg/mL of pIC overnight, before pulsing with OVA in the presence of pIC, and cocultured with B3Z hybridoma cells. IL2 secretion was measured as a read-out for antigen cross-presentation capacity. Results from one experiment involving 6 mice/ group. Data are represented as mean ± SEM over time. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 as compared with PBS-treated tumors. ###, P < 0.001; ####, P < 0.0001 as compared with WT DCs. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Sidak multiple comparisons test) as compared with untargeted control group. ####, P < 0.0001 (two-way ANOVA test for multiple comparisons) as compared with Fpr1−/−-untreated de-iniDCs. D, Immunocompetent C57BL/6 mice were inoculated s.c. with WT murine MCA205 cells. When tumor became palpable, mice received i.t. injection of antigen-pulsed de-iniDC_Cas9 DCs (PBS, as WT control) or Fpr1−/− de-iniDC_Cas9 DCs every 4 days (Fpr1−/− DCs). In addition, 50 μg/mice pIC or an equivalent volume of PBS was i.p. injected on the same day, 1 day, 4 days, 1 week, and 2 weeks after de-iniDC administrations. Data are represented as mean ± SEM over time (n = 6 per group). **, P < 0.01; ****, P < 0.0001 as compared with PBS. ####, P < 0.0001 as compared with Fpr1−/− DCs.

Article Snippet: WT and Fpr1−/− mice were sacrificed, and tumors were harvested and placed in ice-cold RPMI (1 mL) in GentleMACS C tubes (Miltenyi Biotec).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Transfection, Control, Injection

Journal: Cancer Discovery

Article Title: A TLR3 Ligand Reestablishes Chemotherapeutic Responses in the Context of FPR1 Deficiency

doi: 10.1158/2159-8290.cd-20-0465

Figure Lengend Snippet: Figure 3. pIC-based immunotherapy restores the efficacy of chemotherapy in defective Anxa1/Fpr1 signaling in transplanted fibrosarcomas (in a T-cell–dependent manner). WT (A–C) or Anxa1−/− (D) murine MCA205 cells were inoculated s.c. into immunocompetent C57BL/6 WT (A, B, and D) or Fpr1−/− (C) mice. When tumor became palpable, mice received 5.17 mg/kg i.p. MTX, 50 mg/kg i.p. CTX, 50 μg/mice i.p. pIC (injected at days 1, 4, and 7 after the other treatments), or an equivalent volume of PBS, and tumor growth was routinely assessed. In addition, for antibody-mediated depletion experiments, 100 μg of isotype control (Iso), or monoclonal anti-CD4 (αCD4) or anti-CD8 (αCD8) antibodies in 100 μL PBS were injected i.p. 2 days before, on the same day, and 1 week after chemotherapy. In A, C, and D, ****, P < 0.0001, as compared with PBS-treated tumors. ####, P < 0.0001, as compared with MTX-treated tumors. In B, *, P < 0.05; **, P < 0.01; ****, P < 0.0001, as compared with αCD4αCD8. $$$$, P < 0.0001, as compared with MTX+αCD4αCD8. ##, P < 0.01, as compared with MTX+CTX+pIC+αCD4αCD8. Results from one representative experiment out of three independent ones involving at least 5 mice/group and yielding similar results are illustrated. The three plots show negative and positive controls both for chemo-based and chemo-immuno combination therapy. Data are represented as mean ± SEM over time.

Article Snippet: WT and Fpr1−/− mice were sacrificed, and tumors were harvested and placed in ice-cold RPMI (1 mL) in GentleMACS C tubes (Miltenyi Biotec).

Techniques: Injection, Control

Journal: Cancer Discovery

Article Title: A TLR3 Ligand Reestablishes Chemotherapeutic Responses in the Context of FPR1 Deficiency

doi: 10.1158/2159-8290.cd-20-0465

Figure Lengend Snippet: Figure 7. Effect of pIC chemo-immuno combination therapy on hormone-induced breast cancers. A, After treatment, tumor growth (left) was routinely assessed, and results from two pooled independent experiments involving 5 mice/group and yielding similar results are illustrated. Data are represented as mean ± SEM over time. Kaplan–Meier curves (right) with death or tumor size exceeding 200 mm2 as endpoint are shown. ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, as compared with PBS-treated tumors developed in mice of the same genotype; ###, P < 0.001; ####, P < 0.0001, as compared with MTX+CTX-treated tumors developed in mice of the same genotype; $$$, P < 0.001; $$$$, P < 0.0001, as compared with pIC-treated tumors developed in mice of the same genotype. Individual growth curves from WT (B) and Fpr1−/− (C) mice belonging to the experiment depicted in A. From left to right mice injected with (i) PBS or pIC; (ii) PBS or MTX combined with CTX; (iii) PBS or MTX combined both with CTX and pIC; and (iv) MTX + CTX or MTX + CTX also combined with pIC.

Article Snippet: WT and Fpr1−/− mice were sacrificed, and tumors were harvested and placed in ice-cold RPMI (1 mL) in GentleMACS C tubes (Miltenyi Biotec).

Techniques: Injection