A fluorescence digital image microscopy system for quantifying relative cell numbers in tissue culture plates

Abstract

Microwell tissue culture plates provide a convenient format for conducting cell growth and cellular cytotoxicity assays, but such assays often have a limited dynamic range. We have developed the digital imaging microscopy scanning system (DIM‐SCAN), which is a semiautomated fluorescence digital imaging system for quantifying relative cell numbers in situ in a variety of tissue culture plate formats, especially 96‐well plates. The system consists of an epifluorescence inverted microscope with a motorized stage, video camera, image intensifier, and an 80386 microcomputer with a PC‐Vision digitizer. Turbo Pascal software controls the stage and scans the plate taking multiple images per well, calculates total fluorescence per well, provides for daily calibration, and configures easily for a variety of tissue culture plate formats. Thresholding of digital images is used to remove background fluorescence without rinsing. Using cells pretreated with fluorescein diacetate (FDA) and loaded into 96‐well plates, linearity was seen over 3.0 logs of cell density (r = 0.996), with similar results in 6‐well (r = 0.972), 24‐well (r = 0.970), and 48‐well plates (r = 0.981). For cells stained directly in plates with Hoechst 33342, linearity was observed over 2.7 logs of cell density (r = 0.975). In low‐viability cultures stained with FDA in 96‐well plates, excessive background fluorescence completely masked viable cells, but digital thresholding dramatically reduced background fluorescence, producing a linear response (r = 9.333) over 2.7 logs of cell density (from 264 cells/well to 1.35 × 105 cells/well). DIM‐SCAN is a versatile system for quantifying total or viable cell numbers in tissue culture plates over a wide dynamic range. © 1996 Wiley‐Liss, Inc.

Human NKT Cells Mediate Antitumor Cytotoxicity Directly by Recognizing Target Cell CD1d with Bound Ligand or Indirectly by Producing IL-2 to Activate NK Cells

“…Tumor cell viability was measured by quantifying retained calcein-AM fluorescence with digital image microscopy scanning (DIMSCAN), as previously described (35,36). Calcein-AM (Molecular Probes, Eugene, OR) was added to the target cells, 5 g/ml, and the cells were incubated at 37°C for 30 min.…”

Antidisialoganglioside/granulocyte macrophage–colony-stimulating factor fusion protein facilitates neutrophil antibody-dependent cellular cytotoxicity and depends on FcγRII (CD32) and Mac-1 (CD11b/CD18) for enhanced effector cell adhesion and azurophil granule exocytosis

“…23,24 Neuroblastoma cells were detached with Puck saline A and 1 mM EDTA (Puck EDTA), 25 washed, and resuspended in complete RPMI 1640 medium. Calcein-AM was added to produce a final concentration of 5 g/mL, and cells were incubated at 37°C for 30 minutes in the dark.…”

Synergistic cytotoxicity of buthionine sulfoximine (BSO) and intensive melphalan (L-PAM) for neuroblastoma cell lines established at relapse after myeloablative therapy

“…26 Following incubation with drugs or control medium, a vital stain, fluorescein diacetate (10 mg/ml) was added to the 96-well plate and incubated for 20 min. Eosin-Y (800 mg/ml) was then added to quench background fluorescence in the medium and in non-viable cells.…”

“…The plates were then analyzed on an inverted microscope with the relative fluorescence (RF) of each well quantitated by the DIMSCAN digital imaging system software, using digital thresholding to eliminate background fluorescence. 26 The mean fluorescence for treated wells was compared to control wells to derive the surviving fraction. DIMSCAN is a new method developed in our laboratory that is superior to other cytotoxicity assays in 96-well plates by virtue of its ability to measure greater than four logs of cell killing.…”

“…Comparison studies have shown that viable cells measured by DIMSCAN exclude trypan blue, and are clonogenic. 26,27 Furthermore, drug sensitivity/resistance measured by DIMSCAN correlates with clinical drug exposure. 28,29 …”