U 100 insulin syringe

All procedures were approved by the Regional Commission of Freiburg, Baden - Württemberg. Approximately 10⁷colony forming units (CFUs) S. aureus in 10 µl PBS were intradermally (i.d.) injected (30 gauge needle, U-100 insulin syringe (BD)) into the ear pinna of anaesthetized mice. In some experiments, heat fixed (hf) bacteria were used for i.d. inoculation (10⁸ hf bacteria in 10 µl PBS per ear pinna). For re-infection experiments either 10⁷ CFU S. aureus in 10 µl PBS or 10⁸ hf bacteria in 10 µl PBS were i.d. injected into the same ear pinna 3 weeks (if not indicated otherwise) after the first infection. If not indicated otherwise groups of 4–5 mice were used per experiment followed, by at least one repetition to confirm the results. Lesion morphology was documented by digital photographs (Canon PowerShot A650 IS) of mice ears and analyzed by the software program Adobe Photoshop CS6.

When the ongoing (unstimulated) neuronal activity of a unit was visibly stable, it was recorded for a control period of 30 min without any treatment (baseline activity). Subsequently, saline (0.9 % NaCl, 50 μl) was injected with a U-100 insulin syringe (BD, Franklin Lakes, NJ, USA) deep into the temporal muscle close to the crista temporalis within one minute. After another 30 min of recording, 50 μl of 10 mM α,β-meATP (α,β-methylene adenosine 5′-triphosphate disodium salt hydrate, Sigma-Aldrich, Taufkirchen, Germany) was injected into the temporal muscle and the recording continued for 60 min. After another 10 min without further treatment (new baseline), in most of the experiments 60 μl of 2 % lidocaine (Xylocain, Astra Zeneca, Wedel, Germany) was injected into the ipsilateral medial (semispinalis and rectus capitis) neck muscles followed by a recording period of 20 min. Next the temporal muscle was injected with 60 μl lidocaine and the activity recorded for another 20 min. Finally, 60 μl of lidocaine was applied onto the exposed cranial dura mater followed by a final recording period of 20 min. In some experiments 60 μl of lidocaine was injected only into the ipsilateral temporal muscle immediately followed by 50 μl of α,β-meATP, then the recording was continued for 60 min.

C57BL/6J mice (4 and 22 months old) were purchased from Harlan NIA as part of the age-controlled NIH mouse colony program. All animal procedures were carried out according to a protocol approved by the Institutional Animal Care of Albert Einstein College of Medicine. In some experiments, mice were injected in the footpad of both legs with tree different pathogens: a nonvirulent strain of C. neoformans (H99), stained with 1% Uvitex 2B, with M. smegmatis expressing GFP, and with S. aureus (Wood strain without protein A) BioParticles®, Alexa Fluor® 488 conjugate (Molecular Probes®, San Diego, CA, USA).
An amount between 1 × 10⁷ was injected in each leg with a 3/10 mL BD Lo-Dose™ (Franklin Lakes, NJ, USA) U-100 insulin syringe with 29 G × 1/2 in. BD Ultra-Fine™ (Franklin Lakes, NJ, USA) IV permanently attached needle. After different time points (5 min, 20 min, 1 h, and 2 h), the popliteal lymph node (PLN) and collector running from the injection site to the PLN were collected. The derma of the injection site was collected after 5 min as well.

Tamoxifen (TAM) was administered by subcutaneous (s.c.) injections (BD Micro-Fine 0.5 mL; 29G; U-100 Insulin Syringe; REF 324824) up to three times between postnatal (P) days 4 and 14 with a maximum of one dose per day. Mice were induced with 100 mg/kg body weight TAM (Sigma-Aldrich; T5648) dissolved in corn oil (Sigma-Aldrich; C8267). As negative controls, corn oil-induced PDGFRβ-P2A-CreER^T2-tdTomato mice were used.

Nude mice (8 weeks of age) or WT and TNCKO (C57Bl/6J) mice (bred in house) were grafted in the first third part of the tongue. In particular, 3 × 10⁶ OSCC13 cells in 10 µl PBS were injected using a U-100 insulin syringe (BD Micro-Fine) in C57Bl/6J mice. Some 1 × 10⁶ cells OSCC13-LUC (luciferase expression vector expressing cells) were similarly grafted. Tumors were visible 2 weeks upon engraftment and mice were sacrificed for analysis at 3 weeks (bioluminescence experiment), 2 and 4 weeks (NIR) or 4 weeks (IR). For irradiation analysis, a 2 Gy unique dose of photons was delivered two weeks after tumor cell engraftment (3 × 10⁶ cells). Mice were sacrificed two weeks later and tissue was analyzed by staining. For bioluminescence detection, a RediJect D-Luciferin Ultra Bioluminescent Substrate (Perkinelmer 770505) solution at 30 mg/ml was injected intraperitoneally 7 min before imaging. Images were acquired for 5 min using a live imager (NightOwl, Berthold). All mice were housed and handled according to the guidelines of INSERM and the ethical committee of Alsace, France (CREMEAS) (Directive 2010/63/EU, 01386.02, C-67-482-033 on the protection of animals used for scientific purposes).

A U-100 insulin syringe equipped with a 29-gauge needle (BD) was used to inject 10 μL aliquots of a 1 × 10⁶ cfu mL⁻¹ bacterial inoculum in PBS or 10 mM MgCl₂ into the haemocoel of each G. mellonella larvae via the last left proleg. Before and after injection, the area was cleaned using an alcohol swab. After injection, G. mellonella larvae were incubated in plastic containers and kept at room temperature in the dark. The number of dead larvae were scored daily for 72 h. Larvae were considered dead when they displayed no movement in response to a stab to the head. The crude cell-free supernatant (derived by centrifuging and filtering (0.2 μM)) from an overnight (O/N) broth culture of the bacterial strains was also used to assess toxicity.