recombinant human epidermal growth factor  (R&D Systems)

Journal: STAR Protocols

Article Title: Protocol for CRISPR-Cas9-mediated induction of KMT2A rearrangements in cell line and umbilical cord blood hematopoietic stem and progenitor cells

doi: 10.1016/j.xpro.2024.103481

Figure Lengend Snippet:

Article Snippet: Human hematopoietic stem cell expansion cytokine package (IL-6) (100 μg SCF, 100 μg FLT3 ligand, 100 μg TPO, 20 mg IL-6) , PeproTech , Cat# HHSC6.

Techniques: Cell Culture, Recombinant, Red Blood Cell Lysis, Reverse Transcription, TA Cloning, Software

Journal: STAR Protocols

Article Title: Protocol for CRISPR-Cas9-mediated induction of KMT2A rearrangements in cell line and umbilical cord blood hematopoietic stem and progenitor cells

doi: 10.1016/j.xpro.2024.103481

Figure Lengend Snippet: Medium for UCB HSPCs

Article Snippet: Human hematopoietic stem cell expansion cytokine package (IL-6) (100 μg SCF, 100 μg FLT3 ligand, 100 μg TPO, 20 mg IL-6) , PeproTech , Cat# HHSC6.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer

doi: 10.3390/ijms25158085

Figure Lengend Snippet: Preparation of the anakinra-loaded sphingomyelin nanosystems (ANK-SNs). ( A ) Schematic representation of the single-step SN preparation by the ethanol injection method, followed by the dropwise addition of ANK solution in PBS at physiological pH. ( B ) ANK isolation from its pharmaceutical form (Kineret ® ) by using desalting columns. (Created with BioRender.com ).

Article Snippet: The next day, the medium was replaced with IMDM 2% FBS alone or with 20 ng/mL of each recombinant human IL-1α + IL-1β (R&D Systems, Minneapolis, MN, USA).

Techniques: Injection, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer

doi: 10.3390/ijms25158085

Figure Lengend Snippet: Physicochemical characterization of sphingomyelin nanosystems (SNs) (VitE/SM/NHS) and anakinra-loaded sphingomyelin nanosystems (ANK-SNs) (VitE/SM/NHS/ANK) using several analytical techniques. ( A ) Size (nm) (black columns) and surface charge (mV) (turquoise columns) of SNs and ANK-SNs, measured by dynamic light scattering (DLS) and dynamic light scattering (LDA). Data are expressed as mean ± SD (at least n = 5). ( B ) Complementary physicochemical characterization by nanoparticle tracking analysis (NTA) of SNs and ANK-SNs (n ± 5) is represented as size vs. light scattering intensity (arbitrary unit a.u.). ( C ) Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) analysis of native ANK (calculated exact mass of 17.260 kDa) and ANK-SNs (calculated mean mass ~20 kDa). ( D ) Representative field emission scanning electron microscopy (FESEM) images of SNs (left) and ANK-SNs (right). SN scale bar: 200 nm. ANK-SN scale bar: 1 μm.

Article Snippet: The next day, the medium was replaced with IMDM 2% FBS alone or with 20 ng/mL of each recombinant human IL-1α + IL-1β (R&D Systems, Minneapolis, MN, USA).

Techniques: Electron Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer

doi: 10.3390/ijms25158085

Figure Lengend Snippet: Internalization and cytotoxicity assays of nanosystems using pancreatic ductal adenocarcinoma (PDAC) cells. ( A ) Representative confocal microscopy images after 2 h and 4 h treatment in L3.6p cells. Fluorescent-labeled TopFluor ® sphingomyelin nanosystems (SNs) and TopFluor ® anakinra-loaded sphingomyelin nanosystems (ANK-SNs) are in green. Cell nuclei stained with Hoechst 33342 are in blue. ( B ) Cell viability determined by Alamar Blue TM assay after treatment with SNs and ANK-SNs (0.1 mg/mL to 10 mg/mL) for 4 h and 24 h. Data are expressed as means ± SD. The dotted line is set at 50% of viability.

Article Snippet: The next day, the medium was replaced with IMDM 2% FBS alone or with 20 ng/mL of each recombinant human IL-1α + IL-1β (R&D Systems, Minneapolis, MN, USA).

Techniques: Confocal Microscopy, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer

doi: 10.3390/ijms25158085

Figure Lengend Snippet: Anakinra (ANK) and anakinra-loaded sphingomyelin nanosystems (ANK-SNs) are equivalent in down-modulating cytokine secretion by IL-1-activated cancer-associated fibroblasts (CAFs). ( A ) In vitro experimental model. CAFs were treated with recombinant IL-1α + IL-1β, mimicking tumor-derived IL-1, in the absence or in the presence of ANK, ANK-SNs, or SNs. Secretion of TSLP, IL-8, IL-6, and TGF-β was measured after culture. (Created with BioRender.com ). ( B ) Dose–response curve to detect the best concentrations of ANK for inhibiting TSLP secretion by CAFs (n = 3). ANK was added at the indicated concentrations. Untreated CAFs were used as negative (nt) control; IL-1α + IL-1β-treated CAFs were used as positive (IL-1α + IL-1β) control. ( C – F ) ANK, ANK-SNs, and SNs (VitE/SM/NHS formulation), tested for their capacity to down-modulate cytokine secretion, were added at the indicated concentrations and based on the titration curve shown in ( B ). Negative and positive controls used were as in B. ( C ) TSLP. Left , TSLP secretion with controls (n = 2). Right , TSLP percentage inhibition of cumulative experiments (n = 7). ( D ) IL-8. Left , IL-8 secretion with controls (n = 2). Right , IL-8 percentage inhibition of cumulative experiments (n = 6). ( E ) IL-6. Left , IL-6 secretion with controls (n = 2) . Right , IL-6 percentage inhibition of cumulative experiments (n = 6). ( F ) TGF-β. Left , TGF-β secretion with controls (n = 2). Right , TGF-β percentage inhibition of cumulative experiments (n = 3). Data are mean ± SEM from the indicated number (n) of independent experiments. Significance was calculated by one-way ANOVA test and Newman–Keuls post-test. Values were considered significantly different for * p < 0.05, ** p < 0.01, *** p < 0.001; ns = not significant.

Article Snippet: The next day, the medium was replaced with IMDM 2% FBS alone or with 20 ng/mL of each recombinant human IL-1α + IL-1β (R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Recombinant, Derivative Assay, Control, Formulation, Titration, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer

doi: 10.3390/ijms25158085

Figure Lengend Snippet: Anakinra-loaded sphingomyelin nanosystems (ANK-SNs) are superior to free anakinra (ANK) in down-modulating the secretion of IL-17, but not of IFN-γ, by in vitro differentiated Th17 cells. ( A ) In vitro experimental model. Naïve CD4 + T cells were differentiated towards Th17 cells by treatment with anti-CD2/CD3/CD28-coated beads and IL-1β, IL-23, IL-6, and TGF-β, and in the absence or in the presence of ANK, ANK-SNs, or SNs. On day 5, Th17 cells were collected and restimulated with anti-CD2/CD3/CD28-coated beads, and IL-17 and IFN-γ secretion levels were measured by ELISA. (Created with BioRender.com ). ( B ) Dose–response curve to determine the best concentrations of ANK for inhibiting IL-17 secretion in Th17 cells (n = 3). ANK was added at the indicated concentrations. Th0 (i.e., naïve CD4 + T cells activated with anti-CD2/CD3/CD28-coated beads only) were used as a negative control. Th17 cells were used as a positive control. ( C , D ) ANK, ANK-SNs, and SNs (VitE/SM/NHS formulation), tested for their capacity to down-modulate cytokine secretion by Th17 cells, were added at the indicated concentrations and based on the titration curve shown in B. ( C ) IL-17. Left , IL-17 secretion with controls (n = 2). Right , IL-17 percentage inhibition of cumulative experiments (n = 11) ( D ) IFN-γ. Left , IFN-γ secretion with controls (n = 2). Right , IFN-γ percentage inhibition of cumulative experiments (n = 10). ( E ) IL-17 percentage inhibition of cumulative experiments using 5 μg/mL of ANK/ANK-SNs (n = 6). ( F ) IFN-γ percentage inhibition of cumulative experiments using 5 μg/mL of ANK/ANK-SNs (n = 4). Data are mean ± SEM from the indicated number (n) of independent experiments. Significance was calculated by one-way ANOVA test and Newman–Keuls post-test. Values were considered significantly different for * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The next day, the medium was replaced with IMDM 2% FBS alone or with 20 ng/mL of each recombinant human IL-1α + IL-1β (R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control, Formulation, Titration, Inhibition