Here we show how to determine the total number of individualized viable moss cells per well. The method of choice is the measurement of the autofluorescence, represented by fluorescence from chlorophyll. With the LVF monochromator™ of the CLARIOstar® microplate reader moss cells down to 500 cells per well can be detected. Optimized instrument settings were found after well scanning pretests and include the use of the orbital averaging function to cover unevenly distributed protoplasts.
The number of viable cells is compared to the relative number of freshly transformed, GFP-expressing moss cells. The data show that excitation of wild type GFP at 385 nm will also excite chlorophyll. Therefore, for analysis of GFP expressing moss cells, it is necessary to use non-transformed cells as background control.
The measurements were done in cooperation with the company Greenovation Biopharmaceuticals, an expert in developing highly efficient biopharmaceuticals in plants.