Aria 2 system

Transfected HeLa cells were exposed to blue light (50 μmol m⁻² s⁻¹) for 3 h. The cells were then labeled with Alexa Fluor 488 annexin V and propidium iodide (PI) according to the protocol of the Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (V13241, Invitrogen) for flow cytometry. The labeled cells were analyzed using the BD Aria II system. The relative cell death rate was calculated as the number of apoptotic cells in blue light divided by the number of apoptotic cells in the dark. For FAM-LETD-FMK caspase-8 assay in Hela cells using flow cytometry, the transfected HeLa cells were exposed to blue light (30 μmol m⁻² s⁻¹) for 12 h, stained with the Image-iT LIVE Green Caspase-8 Detection Kit (I35105, Invitrogen) then the labeled cells were analyzed using the BD Aria II system.

BM cells were isolated, lineage depleted, and stained as described above. CD45.2⁺ MPs (Lin^-cKit⁺ScaI⁻), MDPs, or CDPs were isolated from whole BM of tumor-bearing or tumor-free mice by cell sorting on the ARIAII system (BD). A total of 2500 sorted progenitors were plated on 1.125 × 10⁶ CD45.1⁺ BM cell feeder culture in RPMI-1640 medium (Lonza) supplemented with 10% fetal bovine serum (Atlanta Biological), β-mercaptoethanol (Gibco), non-essential amino acids (Life Technologies), and l-glutamine (Life Technologies) in the presence of 100 ng/ml recombinant Flt3L and/or 100 ng/ml recombinant GCSF (PeproTech) in 24-well plates. The medium was replaced after 3 days and cultures were analyzed after 5 days. To lift cells, 0.05% Trypsin (HyClone) was used. Cells were stained and analyzed as described for flow cytometry, identifying the progeny of isolated progenitors by CD45.2 positivity. For GCSF priming experiment, progenitors were treated with 100 ng/ml GCSF or media alone for 24 h prior to plating in differentiation assay described above.