Optimized digital counting colonies of clonogenic assays using ImageJ software and customized macros: Comparison with manual counting

Abstract

Purpose: To develop a digital method for counting colonies that highly replicates manual counting.

Materials and methods: Breast cancer cells were treated with trastuzumab-conjugated gold nanoparticles in combination with X-ray irradiation, ¹¹¹In labeled trastuzumab, or γ-radiation, followed by clonogenic assays. Colonies were counted manually or digitally using ImageJ software with customized macros. Key parameters, intensity threshold and minimum colony size, were optimized based on three preliminary manual counts or blindly chosen. The correlation of digital and manual counting and inter- and intra-experimenter variability were examined by linear regression. Survival curves derived from digital and manual counts were compared by F-test (P < 0.05).

Results: Using optimized parameters, digital counts corresponded linearly to manual counts with slope (S) and R² value close to 1 and a small y-intercept (y₀): SK-BR-3 (S = 0.96 ± 0.02, R² = 0.969, y₀ = 5.9 ± 2.2), MCF-7/HER2-18 (S = 0.98 ± 0.03, R² = 0.952, y₀ = 0.74 ± 0.47), and MDA-MB-231 cells (S = 1.00 ± 0.02, R² = 0.995, y₀ = 3.3 ± 4.5). Both reproducibility and repeatability of digital counts were better than the manual method. Survival curves generated from digital and manual counts were not significantly different; P-values were 0.3646 for SK-BR-3 cells and 0.1818 for MCF-7/HER2-18 cells. Using blind parameters, survival curves generated by both methods showed some differences: P-values were 0.0897 for SK-BR-3 cells and 0.0024 for MCF-7/HER2-18 cells.

Conclusions: The colony counting using ImageJ and customized macros with optimized parameters was a reliable method for quantifying the number of colonies.

Keywords::

• Clonogenic assay
• ImageJ
• breast cancer cells
• γ-rays

Acknowledgements

This research was supported by grants (# 019374 and # 019513) from the Canadian Breast Cancer Research Alliance. NC is supported by a Vanier Canada Graduate Scholarship from the Canadian Institute of Health Research, a pre-doctoral fellowship from the U.S Army Department of Defense Breast Cancer Research Program (W81XWH-08-1-0519, P00002), and a pre-doctoral fellowship from the Connaught Fund, University of Toronto. The authors thank Ms D. Scollard for editing this manuscript.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Appendix

Macro files for images acquired using FluorChem^TR gel documentation system

Colony_pro.txt

run(‘Images to Stack’);

run(‘Z Project…’, ‘start = 1 stop = 3 projection = [Average Intensity]’);

run(‘Subtract Background…’);

Make_oval.txt

makeOval(120, 591, 419, 416);

Make_rectangle.txt

makeRectangle(44, 42, 1117, 646);

Count_colony.txt

run(‘Set Measurements…’, ‘area mean standard min center integrated limit display redirect = None decimal = 3’);

resetThreshold();

//run(‘Threshold…’);

setAutoThreshold();

setThreshold(0, z1);

run(‘Analyze Particles…’, ‘size = z2-50000 circularity = 0–1.00 show = Masks display clear summarize record’);

run(‘Watershed’);

run(‘Analyze Particles…’, ‘size = z2-500 circularity = 0.30–1.00 show = Masks display clear summarize record’);

resetThreshold();

Macro files for images acquired using a desktop scanner (Dell Photo A10 Printer 922)

Count_scanner1.txt

run(‘Subtract Background…’, ‘rolling = 15 light’);

run(‘8-bit’);

Make_ovalscan.txt

makeOval(111, 1181, 846, 825);

Count_scanner2.txt

setThreshold(0, z1);

run(‘Set Measurements…’, ‘area mean standard min center perimeter integrated display redirect = None decimal = 3’);

run(‘Analyze Particles…’, ‘size = z2-50000 circularity = 0.00–1.00 show = Masks display clear summarize record’);

run(‘Watershed’);

run(‘Analyze Particles…’, ‘size = z2-500 circularity = 0.2–1.00 show = Masks display clear summarize record’);

Note: z1 in ‘setThreshold(0, z1)’; and z2 in run(‘Analyze Particles…”, ‘size = z2-…’ are adjustable parameters and should be chosen based on three manual colony counts of three wells or flasks.