Nexcelom cellometer auto t4 cell counter

Nasal lavage fluid was diluted with 3 ml of complete DMEM and oropharyngeal swabs were collected from the distal oropharynx and immediately placed in 2 ml of complete tissue culture medium in 15 ml polypropylene tubes on ice. To dislodge cells from the swabs, the samples were lightly vortexed. Fluid was subsequently transferred to a new tube and the swabs were again rinsed with 2 ml of PBS and pooled with the first wash. Pooled samples were filtered through a 70 μm cell strainer (CellTreat, Pepperell, MA) to remove large debris and mucus. Samples were then centrifuged and the pellets were resuspended in 0.5 ml of PBS and 10 μl aliquots were stained with 0.4% trypan blue and blue-excluding cells were counted using a Nexcelom Cellometer™ Auto T4 cell counter (Nexcelom; Lawrence, MA). Nasal lavage samples were processed similarly. To determine cell types and activation states, equivalent numbers of cells were processed and stained for flow cytometric analyses as described above.