Safety multifly needle

To avoid contamination, 50% ethanol was used to clean fertilized white Lohmann chicken eggs. At a temperature of 37.5 °C and a humidity of approximately 60–70%, the eggs were positioned with an upright orientation in an incubator to induce embryogenesis (Bruja 3000 digital, Siepmann, Germany and Mini Pro 147, Maino, Italy). After optimization of our protocols, as discussed in our previously published studies, the CAM was lowered by removing 4–8 mL albumin on ED 5 with a 10 mL sterile syringe and a 20-gauge safety butterfly cannula (Safety-Multifly Needle, Sarstedt, Nümbrecht, Germany). A surgical tape was used to reseal the puncture site (3M Micropore surgical tape, Saint Paul, MN, USA). Under aseptic conditions, a window was carefully opened in the eggshell on ED 6 without damaging the underlying embryonic structures to facilitate the implantation of the generated tumor cell pellets. Subsequently, Parafilm was placed over the opening (Bemis Company Inc., Neenah, WI, USA) and the eggs were positioned back in the incubator until the implantation on ED 7.

Briefly, fertilized white Lohmann chicken eggs were cleaned with 50% ethanol to avoid potential contamination. The eggs were placed in an upright position in an incubator at a temperature of 37.5 °C and a humidity of approximately 60–70% to induce embryogenesis (Bruja 3000 digital, Siepmann, Germany and Mini Pro 147, Maino, Italy). On day five of embryonic development, the CAM was lowered by removing 4–6 or 8–10 mL albumin with a 10 mL sterile syringe and an 20-gauge safety butterfly cannula (Safety-Multifly Needle, Sarstedt, Nümbrecht, Germany). The puncture entrance was resealed with surgical tape (3M Micropore surgical tape, Saint Paul, MN, USA). On experimental day 6 (ED6), a window was cut in the shell of the eggs under aseptic conditions, exposing the embryonic structures. The aperture of the eggshell was subsequently covered with Parafilm (Bemis Company Inc., Neenah, WI, USA).

The following parameters were measured using multiplex ELISA (Myriad RBM, Austin, Texas) in stimulated whole-blood cultures: BDNF, CCL2, CCL3, CCL4, CXCL8, CCL11, Factor VII, GM-CSF, ICAM-1, IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, IL-17, IL-18, IL-23, IL-1ra, IL-1α, IL-1β, MMP-3, MMP-9, SCF, TNF-α, TNF-β, and VEGF. Here, 1 mL of whole blood was withdrawn in blood collection tubes (TruCulture, EDI GmbH, Reutlingen, Germany) by Safety-Multifly Needle (Sarstedt, Nümbrecht, Germany). The collection tubes contained the stimulants lipopolysaccharide (LPS) from Escherichia coli serotype O55:B5 and Staphylococcus enterotoxin B (SE-B) in a concentration of 0.1 µg/mL. Each tube was incubated for 24 h in a dry block incubator (VLM GmbH, Bielefeld, Germany) at 37°C. After 24 h, a valve separator was inserted into the tube and the samples were stored at −80°C until further analyses. For more details, see work of our own group [20 (link)]. Additionally, six inflammatory mediators were measured in serum samples using multiplex ELISA: CCL2, CXCL8, IL-1ra, IL1- β, IL-6, and TNF-α.