Materials and Methods
This staining protocol is adapted from a method published by Lin, et al in Frontiers in Plant Science (1). FDA (Sigma-Aldrich cat# F7378) was dissolved in acetone to make 5 mg/mL stock solution. PI (Sigma-Aldrich cat# P4864) was diluted to 0.5 mg/mL in 0.4 M D-mannitol. The working solution was made fresh by adding 10 µL FDA and 10 µL PI in 0.5 mL of 0.4 M D-mannitol. The solution has a finite lifetime of about 30 minutes. Fresh solution should be prepared after 30 minutes. 5 µL of working solution was added to 10 µL of cell solution. The cells were allowed to incubate for 3-4 minutes. The cell solution was gently mixed, and a 10 µL aliquot was dispensed into the CellDrop counting chamber and the cell sample was counted in the green and red fluorescent channels (Figure 1). A cell counting protocol was prepared based on the dilution factor applied and the expected diameter of the protoplasts.
Refer to Tech Note 186 – CellDrop Best Practices for additional guidance on sample loading, proper focus, and proper exposure. See www.denovix.com/celldrop-videos for more information.
Protoplast Protocol Settings
Table 1: Count Settings
|Count Application||AO/PI or Custom|
|Chamber Height||100 µm|
|Diameter (min)||6 µm|
|Diameter (max)||55 µm|
|Green Fluorescence Threshold||1|
|Red Fluorescence Threshold||1|
Results and Summary
The CellDrop reports data for total, live, and dead cell counts in terms of counted cells and cells/mL, as well as mean cell diameter and percent viability when applicable. The CellDrop FL with dual color fluorescence has powerful counting algorithms and customizable count settings to enable accurate counting and viability determination of a wide variety of cell types, including plant protoplasts.
1. V Pinillos & J Cuevas (2008) Standardization of fluorochromatic reaction test to assess pollen viability, Biotechnic & Histochemistry, 83:1, 15-21.
Revised 19 Oct 2020