Nexcelom cellometer auto t4

OT-I Thy1.1⁺ TCR transgenic T cells were harvested from spleen and mLN of naïve animals. Flow cytometry was used to determine the frequency of OT-I T cells prior to adoptive transfer by staining with anti-Vα2 (used by both TCRs) and anti-CD8 (BD Pharmingen, San Diego, CA). Cells were counted with a Nexcelom Cellometer Auto T4 (Nexcelom Bioscience, Lawrence, MA), and 10⁶ WT OT-I T cells with our without the same number of OT-II T cells were transferred into naive C57BL/6 mice i.v. 24 to 48 hours prior to infection or transplantation.

Cells were maintained in DMEM/F12 containing 10% fetal bovine serum under standard tissue culture conditions. Subconfluent cultures in 24-well plates were growth-arrested for 2 days in DMEM/F12 containing 0.4% bovine serum albumin. Next cells were placed in DMEM/F12 containing a low concentration (25 ng/mL) of PDGF-BB and then treated every day for 4 days without or with various treatments. In experiments in which cells were treated with SDF-1α, the SDF-1α was co-administered with sitagliptin (1 μmol/L). Sitagliptin was administered with SDF-1α because we have found that sitagliptin, by blocking DPP4, prevents the metabolic inactivation of SDF-1α and thus enhances its effects on proliferation of cardiac fibroblasts.^{13 (link)} Finally, cells were harvested and cell number quantified using a Nexcelom Cellometer Auto T4 cell counter (Nexcelom Bioscience: Lawrence, MA).