Celltracker orange cmtmr 5 and 6 4 chloromethyl benzoyl amino tetramethylrhodamine

Spleens and lymph nodes were harvested from OT-I.SJL mice and cells incubated with anti-CD8α beads, and T_CD8+ were positively selected using an AutoMACS sorter (Miltenyi Biotech). To assess T_CD8+ proliferation, Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) (Invitrogen) labeled OTI.SJL T_CD8α+ cells were adoptively transferred into mice on day minus 3 by i.v. injection into the tail vein. On day 3, T_CD8+ cell proliferation was determined by dilution of CFDA-SE fluorescence using flow cytometry. For visualization, T_CD8+ were labeled with 5 μM CellTracker Orange CMTMR (5-(and-6)-(4-chloromethyl)benzoyl)amino)tetramethylrhodamine (Invitrogen) and adoptively transferred into mice. Twenty four hours later, the mice were infected with rECTV, and the draining lymph nodes (D-LN) were harvested and frozen. Cryostat sections (30 μm) were cut and fixed in 4% paraformaldehyde.

PreB/pCAGS or preB/FL-RAGE cells were seeded and cultured for the indicated hours before being photographed at 20× magnification in phase contrast with an Apotome microscope (Zeiss, Germany). Aggregates area of 3 different fields was measured with Axiovision Software™ Rel 4.7 (Zeiss). For “mixed” aggregation assays, a double colored assay was performed as already described with same modifications [37] (link), [38] (link). Briefly, preB cells or preB/FL-RAGE (that express GFP) were labelled fluorescent red with 5 µM of CellTracker™ Orange CMTMR (5-)and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (Invitrogen) in RPMI 1640 medium for 45 min at 37°C. After extensive washing, cell lines were cultured as single cell suspensions or mixed in equal number (5×10⁴) as indicated and allowed to aggregate in growth medium for 24 h at 37°C under 5% CO₂. Images were taken using an Apotome microscope (Zeiss, Germany) with a 20× objective and Axiovision Software™ Rel 4.7 (Zeiss).