The establishment of rat basophilic leukemia (RBL) cell lines in the 1970s offered researchers a convenient, if imperfect, model system for the study of difficult-to-obtain mast cells. RBL-2H3 cells subcloned from one of the original RBL cell lines have been used to study the biochemical pathways for secretion in mast cells. Some RBL cell lines are grown as suspension cultures and others are adherent. Because the morphology of the cells under different growth conditions, or even within the same culture, can vary, the ability to create a customized StainFree analysis setting is extremely useful in ensuring accurate cell counts.!
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Figure 1: StainFree cell counts
RBL-1 cells were imaged using the SpectraMax i3 MiniMax 300 Imaging Cytometer, and cells were identified using a custom, user-defined analysis setting. Shown on the left is the original transmitted light image, and on the right is the same image with purple masks indicating cells identified by the software.
Figure 2: StainFree cell counts vs. fluorescent nuclear counts
RBL-1 cells seeded at densities ranging from 156 to 20,000 cells per well were counted using StainFree technology (blue dots), or they were stained with EarlyTox™ Live Red Dye and red fluorescent nuclei were counted (red dots). Cell counts obtained with both methods agreed closely across the entire range of cell densities. Cell counts were measured within a user-defined region of interest (ROI).
Figure 3: FLIPR Calcium 6 Assay on FlexStation 3 Multi-Mode Microplate Reader
RBL-2H3 cells were seeded at 30,000 cells per well in a 96-well plate and incubated overnight. The next day, growth medium was removed, FLIPR® Calcium 6 Assay dye plus probenecid were added to the wells, and cells were incubated for 2 hours. A kinetic fluorescence assay was then performed on a FlexStation® 3 Multi-Mode Microplate Reader. The onboard pipettor delivered the agonist ADP to assay wells, while fluorescent signal indicating calcium mobilization was simultaneously measured from the bottom of the wells by the plate reader.
Figure 4: Fura-2 QBT Calcium assay on FlexStation 3 Multi-Mode Microplate Reader
RBL-2H3 cells were seeded at 30,000 cells per well in a 96-well plate and incubated overnight. The next day they were loaded with Fura-2 QBT™ dye for 60 minutes after removal of growth medium. For IC50 studies, inhibitor YM-58483 was added cells for 45 minutes prior to assay. A kinetic fluorescence assay was then performed on a FlexStation 3 Multi-Mode Microplate Reader. The onboard pipettor delivered CaCl2 to wells at the EC80 concentration (for inhibitor assay, blue dots), or in a titration series (red dots). Fluorescent signal indicating calcium mobilization was simultaneously measured from the bottom of the wells by the plate reader.
Tip :
Since there are often some adherent cells present in suspension cultures of RBL cells, a user-created analysis setting in SoftMax Pro Software can be very helpful in ‘training’ the software to identify only the cells with suspension-type morphology and exclude the attached cells. For adherent cultures, which may vary in their morphology, a user-created analysis setting maximizes identification of all cells regardless of shape.
RBL Cells Analysis Toolkit
- SpectraMax® i3 Multi-Mode Microplate Detection Platform
- SpectraMax® MiniMax™ 300 Imaging Cytometer
- SoftMax® Pro Software
Instrument Settings for imaging and cell counting
| Parameter | Setting for cell counts |
| Optical configuration | SpectraMax MiniMax 300 Imaging Cytometer |
| Read mode | Imaging |
| Read type | Endpoint |
| Wavelength settings | Transmitted light |
| Image acquisition settings | Exposure: 7 ms Focus adjustment: 20 µm |
| Image analysis settings | Analysis type: Discrete Object Analysis Wavelength for finding objects: TL |
| Find objects | Settings: Create new setting |
About StainFree Cell Detection Technology
Imaging cell-based assays typically requires the use of fluorescent probes that can be toxic to living cells or may only function in fixed cells. A label-free method for analyzing cell counts and cell confluence enables researchers to quantitatively monitor cell proliferation and health without time-consuming workflows that may disrupt cell viability.
The SpectraMax i3 Multi-Mode Microplate Platform with MiniMax 300 Imaging Cytometer uses unique, patent-pending StainFree Cell Detection Technology that allows you to perform cell proliferation, cytotoxicity, and other assays without nuclear stains like DAPI, which intercalates with DNA, or live cell dyes that are actually toxic to cells in the long term.