A simple non-perturbing cell migration assay insensitive to proliferation effects

CellTrace dyes distribute consistently through cell divisions such that cells of each generation contain a 2-fold reduction in dye relative to the previous generation.

“The CellTrace dyes have been demonstrated to distribute consistently through cell divisions such that cells of each generation contain a 2-fold reduction in dye relative to the previous generation1718.”

• cites [17] Zhou, W. et al. In Journal of Biological Methods3 (2016).Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [18] Quah, B. J. C. & Parish, C. R. New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes. J Immunol Methods 379, 1–14, 10.1016/j.jim.2012.02.012 (2012). sourcePartly supported

  Section 3.2 (p. 5) reports division profiles for CFSE, CTV and CPD, supporting their use to track successive cell generations. It does not establish a consistent two-fold reduction in dye for every generation; it reports broader peak variance for CTV and CPD, and the Discussion (p. 12) describes dye transfer that increases variability. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

The new method is relatively high throughput in a 96-well format based on a commercial migration platform.

“Our new method is relatively high throughput as we have demonstrated it in a 96-well format based on a commercial migration platform.”

The assay detected inhibition of cell migration resulting from pharmacological destabilization of filamentous actin.

“We also validated that we could detect the inhibition of cell migration resulting from pharmacological destabilization of filamentous actin.”

MCF7 cells were resistant to treatment with cytochalasin D at the commonly used concentration.

“Interestingly, MCF7 cells were resistant to treatment with the actin destabilizing drug, cytochalasin D at the commonly used concentration.”

Cell density did not differ from controls 24 hours after exposure to either dye in any of the cell lines (p = 0.80 for CFR and 0.96 for CV).

“We found that cell density did not differ from controls 24 hours after exposure to either dye in any of the cell lines (p = 0.80 for CFR and 0.96 for CV) (Fig. 2c,d).”

At 1 μg/mL, cytochalasin D inhibited migration in all breast cell lines tested except MCF7.

“At the standard concentration of 1 μg/mL, cytochalasin D inhibited migration in all breast cell lines with the exception of MCF7.”

Under normoxic conditions, there was no discernible difference in migration between cells derived from tumors and apparently normal tissue from the same animal.

“but there was no discernible difference between cells derived from tumors and apparently normal tissue (Fig. 6a,b).”

• Also stated in: § Discussion · plain

  “Interestingly, the behavior of cells isolated from tumors was not different from cells taken from apparently normal tissue from the same animal.”

MMC treatment did not affect motility in the breast cell lines tested.

“MMC treatment, in contrast, did not affect motility in these cell types (Fig. 5c,d).”

Undergraduate students were able to efficiently analyze images after about 30 minutes of instruction.

“We found that undergraduate students were able to efficiently analyze images after about 30 minutes of instruction.”

HeLa cells retained CFR more effectively than CV (p < 0.05).

“HeLa cells had a differential response to the two labels, retaining CFR more effectively than CV (p < 0.05).”

The CellTrace dyes at the tested concentrations did not affect cell migration in any of the four human epithelial cell lines (EPC2, CP-A, HeLa, MDA-MB-231).

“None of the cell lines were affected at the dye concentrations tested (Fig. 2a,b). P-values were 0.87 and 0.99 for CFR and CV, respectively.”

CFR signal was not reduced at 12 hours for any cell type.

“CFR signal was not reduced at 12 hours for any cell type.”

Under normoxic conditions, epithelial cells were much more migratory than fibroblasts in primary cells from the mouse lung cancer model.

“Under standard, normoxic cell culture conditions, we found that epithelial cells were much more migratory than fibroblasts”

• Also stated in: § Discussion · plain

  “We found that generally the epithelial cells were considerably more migratory than the fibroblast.”

The reported assay does not require cell cycle blockade or other perturbing manipulation of normal cell physiology.

“This approach does not require cell cycle blockade or other perturbing manipulation of normal cell physiology.”

The assay was demonstrated on a wide range of cell types including epithelial cells, fibroblasts, human and mouse cells, cancerous and non-cancerous cells of the same lineage, and both immortalized cell lines and primary cells.

“We demonstrated our approach on a wide range of cell types including epithelial cells, fibroblasts, human and mouse cells, cancerous and non-cancerous cells of the same lineage, and both immortalized cell lines and primary cells.”

Hypoxic conditions (1% O2) modestly but significantly reduced migration of both normal and cancer derived epithelial cells.

“We found that hypoxic conditions modestly, but significantly reduced migration of both normal and cancer derived epithelial cells (Fig. 6c,d).”

Inhibition of cell proliferation by pre-incubation with mitomycin C caused an inhibition of migration in some cell types, but not others.

“Significantly, we found that inhibition of cell proliferation by pre-incubation with mitomycin C caused an inhibition of migration in some cell types, but not others.”

For the 12 cell types tested, optimal migration time ranged from 11-24 hours.

“For the 12 cell types that we tested, optimal migration time ranged from 11–24 hours.”

There was no significant difference between the dual image analysis method and the single image method (p = 0.85).

“For the cell lines tested, we found no significant difference between the dual image analysis method and the single image method (p = 0.85).”

Cytochalasin D treatment significantly decreased migration in all epithelial and fibroblast cell lines tested.

“we observed that, as expected, cytochalasin D treatment significantly decreased migration in all cell lines (Fig. 5a,b).”

The assay unambiguously distinguished these cell lines from each other in terms of cell motility with the exception of the two least motile.

“We were able to unambiguously distinguish these cell lines from each other in terms of cell motility with the exception of the two least motile.”

MMC treatment did not affect migration in HeLa and Swiss 3T3 cells.

“In contrast, HeLa and Swiss 3T3 cell migration was unaffected by MMC treatment (Fig. 5b).”

MCF7 was significantly less migratory than both MCF 10A and MDA-MB-231 cells.

“We demonstrated the cancer cell line, MCF7 was significantly less migratory than both MCF 10A and MDA-MB-231 cells.”

MCF7 was the only cell line across all panels tested that did not undergo inhibition with cytochalasin D treatment.

“This was the only cell line across all panels tested that did not undergo inhibition with this treatment.”

The assay measured stimulation of cell migration by use of an ECM-coated substrate and serum treatment.

“We could also measure stimulation of cell migration by use of an ECM-coated substrate and serum treatment.”

The less motile HeLa and fibroblast cells were unaffected by the treatments (growth factor stimulation, collagen coating, or combined).

“The less motile HeLa and fibroblast cells were unaffected by the treatments (Fig. 4c,d).”

The reported assay measures cell migration without the confounding influence of cell proliferation.

“In conclusion, we report here a high throughput, flexible, inexpensive, and simple assay that measures cell migration without the confounding influence of cell proliferation.”

Using the single image variation, the assay labor time is reduced to about 2 hours.

“Using the single image variation of the technique in which a standard set of ROIs is used to analyze multiple post-migration images, the assay labor time is reduced to about 2 hours.”

Fibroblast cells, regardless of tissue origin, were unaffected by oxygen level.

“In contrast, fibroblast cells regardless of tissue origin, were unaffected by oxygen level (Fig. 6e,f).”

The dual and single image analysis methods yielded the same statistical power to discriminate the motility of the cell lines.

“More importantly, the two approaches yielded the same statistical power to discriminate the motility of the cell lines.”

The new method was able to easily discern differences in unstimulated migration between all tested cell lines except the two least motile, HeLa and Swiss 3T3.

“We were able to easily discern differences in unstimulated migration between all cell lines except the two least motile, HeLa and Swiss 3T3 (Fig. 4a,b).”

The assay is unaffected by cell divisions that occur prior to migration or in cells that do not migrate into the exclusion zone.

“The assay is similarly unaffected by cell divisions that occur prior to migration or in cells that do not migrate into the exclusion zone.”

CV and CFR dyes are symmetrically inherited to daughter cells.

“CV and CFR have been shown to evenly distribute to cell progeny at each division1620.”

• cites [1620]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

In an exclusion zone type migration assay, the property of consistent dye distribution allows evaluation of migration in terms of redistribution of the parental cell population without ambiguity due to proliferation.

“In an exclusion zone type migration assay, this property allows evaluation of migration in terms of redistribution of the parental cell population without ambiguity due to proliferation.”

The assay can be multiplexed with other image-based readouts.

“The assay we present can be multiplexed with other, image-based readouts.”

The traditional approach cannot distinguish cells that have physically crawled into the exclusion zone from cells that were born there.

“This traditional approach cannot distinguish cells that have physically crawled into the exclusion zone from cells that were “born” there”

The new approach was validated using three human epithelial cell lines derived from non-cancerous, pre-cancerous, and metastatic cancer tissues, and a mouse fibroblast line.

“We validated the new approach using three human epithelial cell lines derived from non-cancerous, pre-cancerous, and metastatic cancer tissues, and a mouse fibroblast line.”

Our results show that cancer cells do not invariably have increased motility relative to normal cells.

“Our results show that this is not invariably true.”

By labeling cells with a fluorescent lineage tracing dye at the beginning of the assay, migration can be evaluated in terms of the original or parental cell population.

“By labeling cells with a fluorescent lineage tracing dye at the beginning of the assay, migration can be evaluated in terms of the original or parental cell population.”

The described modification to a widely used cell migration assay disambiguates cell migration from cell proliferation.

“We describe here a simple modification to a widely used cell migration assay that disambiguates cell migration from cell proliferation.”

• Also stated in: § Discussion · plain

  “The new strategy presented here circumvents this problem by use of a label that essentially tracks location of the original cell population without regard to proliferation events.”

The reported assay is high throughput.

“In conclusion, we report here a high throughput, flexible, inexpensive, and simple assay that measures cell migration without the confounding influence of cell proliferation.”

Hypoxia stimulated migration in cancer cells is not a ubiquitous phenomenon.

“Our results suggest that hypoxia stimulated migration in cancer cells is not a ubiquitous phenomenon.”

In the majority of cases, cell migration is conflated with cell proliferation in migration assays.

“A significant drawback of these approaches is that in the majority of cases, cell migration is conflated with cell proliferation11.”

• cites [11] Kramer, N. et al. In vitro cell migration and invasion assays Mutat. Res.-Rev. Mutat. Res. 752, 10–24, 10.1016/j.mrrev.2012.08.001 (2013). sourceNot checked — no full text of the source

The lineage-tracing indicator dyes provide useful information on cellular proliferation without additional labeling steps.

“Most notably, without additional labeling steps, the lineage-tracing indicator dyes provide useful information on cellular proliferation.”

The exclusion zone assay relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration.

“A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration.”

• cites [11] Kramer, N. et al. In vitro cell migration and invasion assays Mutat. Res.-Rev. Mutat. Res. 752, 10–24, 10.1016/j.mrrev.2012.08.001 (2013). sourceNot checked — no full text of the source

Because the assay reports cell migration as a ratio of total cells, it is internally controlled for variations in plating density, pre-migration proliferation rate, efficiency of cell attachment, apoptosis, and efficiency of dye uptake.

“Since this assay reports cell migration as a ratio of total cells, it is internally controlled for a wide range of confounding factors including variations in plating density, pre-migration proliferation rate, efficiency of cell attachment, apoptosis, and efficiency of dye uptake.”