28g1 2 needle

Individual groups of 6 week old C57Bl/6 mice (n=3) were anesthetized by 80mg/kg of ketamine and 10mg/kg of xylazine, before being vaccinated with the soluble form of ovalbumin and αGalCer, the microparticles containing ovalbumin and αGalCer, and control PBS by intravenous or sublingual route of injection. Intravenous injection was via the tail vein in a total volume of 100 μl in PBS. Sublingual injection was underneath the mucous membrane in 20 μl of PBS. All injections were performed with 0.5 ml syringe and 28G1/2 needle (Becton Dickinson and Company, Franklin Lakes, NJ).

Bone marrow cells from two to four C57BL/6J mice/genotype were rinsed from the femur and tibia using a disposable LO-DOSE ¹/₂ cc U-100 Insulin Syringe with a permanently attached 28G ¹/₂ needle (Becton Dickinson, Franklin Lakes, NJ). The material was resuspended in RPMI media (ATCC, Manassas, VA) containing 10% fetal bovine serum (ATCC) and grown in T-75 Falcon flasks (BD Falcon, Bedford, MA) until they reached 95% confluency. To remove cells from flasks the monolayer was washed with Dulbecco’s phosphate buffered saline (DPBS, Gibco Invitrogen, Carlsbad, CA) without Ca and Mg, twice and incubated with 0.05% Trypsin (Gibco Invitrogen) for 4 minutes at 37°C. After neutralizing with an equal volume of media and centrifuging at 2,000 rpm for 4 minutes, cells were resuspended and distributed into 4-well culture slides (BD Falcon). Following a 4-hr attachment period, the media was replaced with media containing 0, 10, or 100 μM AZT (Sigma-Aldrich Co, St. Louis, MO) and cells were incubated for 24 hr. To prepare a stock solution, AZT was dissolved in DPBS and the final concentration was calculated using an extinction coefficient of 11,500 m²/mol at an absorbance wavelength of 266 nm.