Celldrop bf

Cell culture was mixed with an equal volume of deciliation solution (1 mM CaCl₂ and 40 mM sodium acetate). Cell density was measured using an automatic cell counter (model R-1, Olympus, or Cell Drop BF, DeNovix). Additionally, the cell size (diameter when a cell is approximated to a sphere) was simultaneously measured using the same cell counter.

A cell counter (CellDrop BF, DeNovix Inc.) was used for qualitative evaluation of particle size within suspensions due to the small sample volume and the fast and simple measurement. A sample volume of 10 μL was always pipetted into the counting chamber. The focus remained set at 710 for each measurement. The instrument is actually recommended for counting cells of a size between 4 and 400 μm. However, since on the one hand the tests carried out here concerned 1 μm particles and on the other hand even agglomerates were not counted correctly, the measurement result was not suitable for quantification. Nevertheless, it was used to optically evaluate the particle size measurements as well as the influences of particle treatment steps.

All six study groups were grown in monolayer culture with a loading density of 5000 cells/cm2 and passaged till 80% confluence. Cell count was performed using a cell counter (Cell Drop BF, De Novix) by the trypan blue exclusion technique. The population doubling time (PDT) for all the groups was calculated using the following formulae:
$$\text{PDT}=\left({\text{log}}_2\text{x}\text{days}\text{in}\text{culture}\right)/\text{log}\left(\text{FD}\right)–\text{log}\left(\text{ID}\right)$$
Where FD: the number of cells obtained at 80% confluence and ID: the initial number of loaded cells. PDT between the groups was compared from passage 1 to passage 2.
DAPI (4’,6-diamidino-2-phenylindole) was used to define the percentage of cells in distinct cell cycle phases. The cells were trypsinised at 60–70% confluence, and an automated cell counter estimated the cell size at passage1-2 (Countess, Invitrogen). The harvested cells were fixed with 70% ice-cold ethanol for 60 min. After a wash, 1μg/ml of DAPI was added for 30 minutes were the cells washed and the cells resuspended for flow cytometry analysis using BD FACS Celesta flow cytometer using BD FACSDiva Software Version 8.0.1.1. Cell cycle analysis was done using Flow-Jo v10.8.1 software with Dean Jett algorithm