In addition brain, liver, lung, spleen and kidneys were mechanically disrupted, passed through a 100 μm cell strainer and plated into 6-well plates.
The single-cell suspension was then filtered through a 40 -µm cell strainer and plated on poly- d-lysine (PDL -coated PDL -coated
After washing, cells were suspended in supplemented DMEM (Gibco), passed through a 70 µm cell strainer, and plated in a T75 flask.
The following day non-adherent cells were removed, filtered with a 70 μm strainer and plated at 150,000 cells/cm in α-MEM plus 10%, hM-CSF 25 ng/ml, 1% glutamine and penicillin/streptomycin 18 U/ml in the absence or presence of RANKL 10 ng/ml.
Dispersed cell mixtures were filtered through a 100 μM nylon cell strainer (BD Biosciences, San Diego, CA), plated on 6-well plates and grown in DMEM media supplemented with 10% fetal bovine serum (FBS) and Endothelial Growth Supplement (Upstate Biotechnology, Charlottesville, VA).
Sentinel paraaortic lymph nodes were washed with PBS, filtered through a 100 μm cell strainer (BD Biosciences), and plated in complete media on tissue culture dishes.
Single-cell suspensions, obtained by cell strainer filtration, were plated in stem-permissive culture medium (serum-free and supplemented with bFGF and EGF).
The cell suspension was filtered through a 40 μm cell strainer (Falcon) and plated in a coated dish for 25 min to allow attachment of non-neuronal cells.
The colony formation efficiency of TL-HMECs transduced with empty vector or vector expressing PVRL4 was compared between unfiltered cell suspensions and cell suspensions that were filtered through a 35 μm nylon mesh strainer prior to plating into methylcellulose.
To obtain mammospheres, cells from monolayer cultures were enzymatically disaggregated (0.05% trypsin EDTA, Gibco) to a single cell suspension, passed though a 40 μm cell strainer (BD Falcon), plated at clonogenic density (2500 cells/cm), and grown in nonadherent culture conditions, as described (Dontu et al, 2003).
