Microtainer sst

To achieve MR silencing in vivo, doxycycline (Dox) was administered by offering food pellets containing 735 mg/kg doxycycline hyclate (Ssniff Spezialdiäten, Soest, Germany; effective concentration: 625 mg/kg Dox) ad libitum to the mice for at least 14 days. Control animals received a standard diet from the same supplier. Blood was collected by heart puncture after having sacrificed the mice, and serum and plasma were separated using Microtainer SST or K2E tubes, respectively (BD Biosciences, Heidelberg, Germany). In order to perform gene expression and histological analyses, individual organs were dissected and either snap-frozen in liquid nitrogen or fixed in formaldehyde solution.

The experimental design is illustrated in Fig. 2a. Stat1^+/+ and Stat1^−/− mice were divided into 3 treatment groups (vehicle, tMWCNT, or rMWCNT) for one and 21 day sample collections. Mice were anesthetized with isoflurane and dosed with 4 mg/kg tMWCNTs (n = 6), 4 mg/kg rMWCNTs (n = 12), or equal volume pluronic vehicle (n = 18) via oropharyngeal aspiration. Half the mice from each treatment group were euthanized via intraperitoneal (i.p.) injection of pentobarbital (Vortech Pharmaceuticals, LTD, Dearborn, MI #NDC 0298–9373-68) at one-day post-exposure and the rest at 21 days post-exposure. Serum was collected immediately and extracted from clotting factors using a BD (Franklin Lakes, NJ) microtainer SST. Two 0.5 mL aliquots of phosphate buffered saline (PBS) were instilled via intratracheal cannulation and retrieved to collect bronchoalveolar lavage fluid (BALF) for cytokine and cellular content. The left lobe of the lung was inflated and fixed for histology with neutral buffered formalin (Azer Scientific, Morgantown, PA #NBF-4-G) and the right lobes were divided equally into RNAlater (Sigma #R0901) for mRNA or snap frozen in liquid nitrogen for protein analysis. Small intestine, heart, spleen, brain, and liver samples were also collected for histology and mRNA analysis.

For tumor-bearing animals, tumors were collected at the experimental endpoints, bisected along the longitudinal axis and fixed in 10% neutral buffered formalin (Sigma). For nontumor-bearing animals, blood was collected from the retro-orbital sinus < 4 h post-treatment on day 17 (experimental endpoint). The serum was isolated (BD Microtainer SST) and stored at − 20 °C. Carcasses were collected at the experimental endpoint, dissected along the midline and fixed in Bouin’s solution (Sigma).

Safety of intraventricular delivery of MSCs was assessed by observing the treated rat for changes in feeding habit, fur, overall behavior, and weight. Rat weight was documented throughout the experiment. Three weeks after MSC intraventricular administration, all rats were anesthetized with isoflurane; blood samples were collected through direct cardiac puncture and 500 µl of the blood was put into the BD Microtainer SST to isolate serum for comprehensive blood chemistry analysis. All remain blood sample was put into BD Vacutainer ACD Solution B tubes to isolate plasma for the cytokines analysis. The rats were perfused with PBS followed by 4% paraformaldehyde in PBS. They were then decapitated, whole brains were dissected, and heart, liver, lungs, spleen, testis, and kidneys were collected. All organs were kept in 4% paraformaldehyde for 24 hours, then washed with PBS twice and soaked in 70% ethanol. Paraffin embedding and sectioning, along with hematoxylin and eosin (H&E) and immunohistochemical staining were performed in the Mayo Clinic Cancer Center Histology Core facility. Cell toxicity evaluation on H&E staining slides were sent out to Vet Path Services, Inc. for an independent pathologist assessment.

Blood was collected from either the saphenous vein for pretreatment time points or via heart puncture at the experimental endpoint. Collections from the saphenous vein utilized capillary tubes (Sarstedt microvette catalog no. CB300 Z), while blood collected via heart puncture utilized a polymer gel-based separator tube (BD Microtainer SST). Following collection, tubes were spun according to the manufacturer’s instructions, and serum was aliquoted and stored at −80°C until use.