30 gauge needle

Colo 205 tumor cells were injected subcutaneously (SC) on the flank (3 × 10⁶ cells in 100 μL) of humanized and non-humanized NSG mice of both genders aged 16–20 weeks, weighing 25–30 g using a 30-gauge needle (Becton Dickson, Rutherford, NJ, USA). Tumor-bearing mice were weighed and randomly divided into six different groups when the tumor volume reached 100 mm³: (1) PBS; (2) Los; (3) Ang (1–7); (4) LNP-TRAIL; (5) Los + LNP-TRAIL; and (6) Ang (1–7) + LNP-TRAIL. The mice were treated with LNP-TRAIL intratumorally every 48 h at a dose of 5 µg of mRNA. Tumor growth was monitored over time using a caliper and IVIS bioluminescence image, as previously reported.40 (link) Tumors were removed 18 and 24 days after transplantation and weighed. The length (L) and width (W) of the tumors were measured and used to calculate tumor volume (V) using the formula V = 0.5 × (L × W²).41 (link)

As previously described,¹⁸ aqueous fluid was collected in ethylenediaminetetraacetic acid-containing capillary tubes (Sarstedt) after corneal paracentesis with a 30-gauge needle (Becton, Dickinson and Company). A volume of 1 to 5 μl of aqueous was collected from each eye, and stored at –80°C in combination with 1X protease inhibitor (Sigma-Aldrich Corp) until assayed. Aqueous protein was quantified using Pierce 660 nm Protein Assay Reagent (Thermo Scientific) for colorimetric detection on the Nanodrop ND-1000 spectrophotometer (Thermo Scientific).
After aqueous collection, the eye was enucleated and frozen on dry ice. The frozen eye was bisected at the limbus. Serum from all animals was collected by cardiac puncture immediately after death. Serum samples were not pooled. The concentration of 32 cytokines was determined using the Milliplex_MAP mouse cytokine/chemokine premixed 32 plex immunology multiplex assay (EMD MilliporeCorp). The cytokines measured were as follows: Eotaxin, CSF1/2/3, IFNγ, IL-1A, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12B, IL-12, IL-13, IL-15, IL-17, CXCL10, CXCL1, LIF, CXCL5, CCL2, CXCL9, CCL3, CCL4, CXCL2, VEGF, TNFα, and CCL5. Samples were analyzed using the MAGPIX system (Luminex) with xPonent software version 4.2 (EMD Millipore). Data analysis was performed using Milliplex Analyst Standard version 5.1 software (EMD Millipore).

In each experimental session, the rats were assigned to either formalin or saline injection group according to their “baseline” PWT50 values to equalize their means as much as possible. Otherwise, a random block design was applied. After acclimatization for ∼30 minutes in an acrylic observation chamber (17 cm × 17 cm × 35 cm) with 3 mirrors to video-capture rat behaviors, 50 µL of 5% formalin solution (Nacalai Tesque Inc, Kyoto, Japan) or an equal volume of saline was injected into the left upper lip of the rat (the upper lip), just lateral to the nose, using a 0.3-mL syringe with a 30-gauge needle (Becton, Dickinson and Company, Franklin Lakes, NJ), and the acute nociceptive behaviors^{20 (link),21 (link),68 (link)} of the rat were video-recorded with a PC for 60 minutes with a web camera (Logicool HD Webcam C525, Logicool Co, Tokyo, Japan). During injection, the rats were held softly with a towel, to which they were acclimatized every day for 1 to 2 weeks during the habituation period, which made anesthetics unnecessary. The duration of time spent in face rubbing behavior was measured postexperimentally by evaluation of the video playback by an evaluator blinded to the rat group. The observation chamber did not have food or water supply.