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Non-invasive and non-destructive measurements of confluence in cultured adherent cell lines.

Busschots S, O'Toole S, O'Leary JJ, Stordal B - MethodsX (2014)

Bottom Line: Many protocols used for measuring the growth of adherent monolayer cells in vitro are invasive, destructive and do not allow for the continued, undisturbed growth of cells within flasks.Images are analysed for confluence using ImageJ freeware resulting in a measure of confluence known as an Area Fraction (AF) output.The average correlation co-efficient from a Spearman correlation (n = 3) was 0.99 ± 0.008 for OVCAR8 (p = 0.01) and 0.99 ± 0.01 for UPN251 (p = 0.01) cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Histopathology, Trinity College Dublin, Central Pathology Laboratory, St. James's Hospital, Dublin 8, Ireland ; Department of Obstetrics and Gynaecology, Trinity College Dublin, Trinity Centre, St James's Hospital, Dublin 8, Ireland.

ABSTRACT
Many protocols used for measuring the growth of adherent monolayer cells in vitro are invasive, destructive and do not allow for the continued, undisturbed growth of cells within flasks. Protocols often use indirect methods for measuring proliferation. Microscopy techniques can analyse cell proliferation in a non-invasive or non-destructive manner but often use expensive equipment and software algorithms. In this method images of cells within flasks are captured by photographing under a standard inverted phase contract light microscope using a digital camera with a camera lens adaptor. Images are analysed for confluence using ImageJ freeware resulting in a measure of confluence known as an Area Fraction (AF) output. An example of the AF method in use on OVCAR8 and UPN251 cell lines is included. •Measurements of confluence from growing adherent cell lines in cell culture flasks is obtained in a non-invasive, non-destructive, label-free manner.•The technique is quick, affordable and eliminates sample manipulation.•The technique provides an objective, consistent measure of when cells reach confluence and is highly correlated to manual counting with a haemocytometer. The average correlation co-efficient from a Spearman correlation (n = 3) was 0.99 ± 0.008 for OVCAR8 (p = 0.01) and 0.99 ± 0.01 for UPN251 (p = 0.01) cell lines.

No MeSH data available.


Related in: MedlinePlus

Tracking cell recovery using AF. Graphs show the recovery of cells over a period of days after carboplatin exposure. The cells received 3-day exposures to carboplatin every 4–5 weeks. Each dose of drug received, denotes a new round of drugging (Round 1–6). The x-axis shows the days after the drug has been removed from the cells and the y-axis gives the AF output at each time point measured. (a) OVCAR8 cells after exposure to 4 μg/ml carboplatin. (b) UPN251 cells after exposure to 2 μg/ml carboplatin.
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fig0025: Tracking cell recovery using AF. Graphs show the recovery of cells over a period of days after carboplatin exposure. The cells received 3-day exposures to carboplatin every 4–5 weeks. Each dose of drug received, denotes a new round of drugging (Round 1–6). The x-axis shows the days after the drug has been removed from the cells and the y-axis gives the AF output at each time point measured. (a) OVCAR8 cells after exposure to 4 μg/ml carboplatin. (b) UPN251 cells after exposure to 2 μg/ml carboplatin.

Mentions: One example where the application of the AF output method is useful is in the development of drug resistant cell models in vitro. AF output can be used to track cell recovery in an objective manner to see consistently when cells have recovered after drug exposure. Cells were plated into T25 flasks at a cell density of 2.6 × 104 cells per flask and drugged with carboplatin (St. James’ Hospital Pharmacy, Dublin, Ireland) on day 2. The doses of carboplatin used were 2 μg/ml for UPN251 and 4 μg/ml for OVCAR8. These doses caused a similar level of death in the cell lines due to different intrinsic resistances to the drug. On day 5 drugged media was removed and replaced with fresh drug free media. Over subsequent days all T25 flasks were examined for confluence using the AF output method. Upon reaching confluence, cells were re-seeded into T75 flasks. Once all cells had recovered, the next round of drugging commenced following the same format as above (provided the cells were 4 weeks after drugging, otherwise drugging was delayed until this time). Fig. 5 shows the recovery of UPN251 and OVCAR8 ovarian cancer cells after carboplatin exposure (2 and 4 μg/ml, respectively) over a number of rounds of drugging during a selection strategy to produce carboplatin-resistant models. The doses of carboplatin that were used, were selected from testing a range of carboplatin doses on each cell line and selecting the dose which exhibited a large amount of cell death followed by a return to logarithmic growth (data not shown). Each cell line received 3-day exposures to carboplatin every 4–5 weeks, with each dose of drug received denoting a new round of drugging (Round 1–6). Generally, as the amount of rounds of drug administration increase the time taken for cells to recover decreases.


Non-invasive and non-destructive measurements of confluence in cultured adherent cell lines.

Busschots S, O'Toole S, O'Leary JJ, Stordal B - MethodsX (2014)

Tracking cell recovery using AF. Graphs show the recovery of cells over a period of days after carboplatin exposure. The cells received 3-day exposures to carboplatin every 4–5 weeks. Each dose of drug received, denotes a new round of drugging (Round 1–6). The x-axis shows the days after the drug has been removed from the cells and the y-axis gives the AF output at each time point measured. (a) OVCAR8 cells after exposure to 4 μg/ml carboplatin. (b) UPN251 cells after exposure to 2 μg/ml carboplatin.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487325&req=5

fig0025: Tracking cell recovery using AF. Graphs show the recovery of cells over a period of days after carboplatin exposure. The cells received 3-day exposures to carboplatin every 4–5 weeks. Each dose of drug received, denotes a new round of drugging (Round 1–6). The x-axis shows the days after the drug has been removed from the cells and the y-axis gives the AF output at each time point measured. (a) OVCAR8 cells after exposure to 4 μg/ml carboplatin. (b) UPN251 cells after exposure to 2 μg/ml carboplatin.
Mentions: One example where the application of the AF output method is useful is in the development of drug resistant cell models in vitro. AF output can be used to track cell recovery in an objective manner to see consistently when cells have recovered after drug exposure. Cells were plated into T25 flasks at a cell density of 2.6 × 104 cells per flask and drugged with carboplatin (St. James’ Hospital Pharmacy, Dublin, Ireland) on day 2. The doses of carboplatin used were 2 μg/ml for UPN251 and 4 μg/ml for OVCAR8. These doses caused a similar level of death in the cell lines due to different intrinsic resistances to the drug. On day 5 drugged media was removed and replaced with fresh drug free media. Over subsequent days all T25 flasks were examined for confluence using the AF output method. Upon reaching confluence, cells were re-seeded into T75 flasks. Once all cells had recovered, the next round of drugging commenced following the same format as above (provided the cells were 4 weeks after drugging, otherwise drugging was delayed until this time). Fig. 5 shows the recovery of UPN251 and OVCAR8 ovarian cancer cells after carboplatin exposure (2 and 4 μg/ml, respectively) over a number of rounds of drugging during a selection strategy to produce carboplatin-resistant models. The doses of carboplatin that were used, were selected from testing a range of carboplatin doses on each cell line and selecting the dose which exhibited a large amount of cell death followed by a return to logarithmic growth (data not shown). Each cell line received 3-day exposures to carboplatin every 4–5 weeks, with each dose of drug received denoting a new round of drugging (Round 1–6). Generally, as the amount of rounds of drug administration increase the time taken for cells to recover decreases.

Bottom Line: Many protocols used for measuring the growth of adherent monolayer cells in vitro are invasive, destructive and do not allow for the continued, undisturbed growth of cells within flasks.Images are analysed for confluence using ImageJ freeware resulting in a measure of confluence known as an Area Fraction (AF) output.The average correlation co-efficient from a Spearman correlation (n = 3) was 0.99 ± 0.008 for OVCAR8 (p = 0.01) and 0.99 ± 0.01 for UPN251 (p = 0.01) cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Histopathology, Trinity College Dublin, Central Pathology Laboratory, St. James's Hospital, Dublin 8, Ireland ; Department of Obstetrics and Gynaecology, Trinity College Dublin, Trinity Centre, St James's Hospital, Dublin 8, Ireland.

ABSTRACT
Many protocols used for measuring the growth of adherent monolayer cells in vitro are invasive, destructive and do not allow for the continued, undisturbed growth of cells within flasks. Protocols often use indirect methods for measuring proliferation. Microscopy techniques can analyse cell proliferation in a non-invasive or non-destructive manner but often use expensive equipment and software algorithms. In this method images of cells within flasks are captured by photographing under a standard inverted phase contract light microscope using a digital camera with a camera lens adaptor. Images are analysed for confluence using ImageJ freeware resulting in a measure of confluence known as an Area Fraction (AF) output. An example of the AF method in use on OVCAR8 and UPN251 cell lines is included. •Measurements of confluence from growing adherent cell lines in cell culture flasks is obtained in a non-invasive, non-destructive, label-free manner.•The technique is quick, affordable and eliminates sample manipulation.•The technique provides an objective, consistent measure of when cells reach confluence and is highly correlated to manual counting with a haemocytometer. The average correlation co-efficient from a Spearman correlation (n = 3) was 0.99 ± 0.008 for OVCAR8 (p = 0.01) and 0.99 ± 0.01 for UPN251 (p = 0.01) cell lines.

No MeSH data available.


Related in: MedlinePlus