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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

Example of images generated from the AF calculation method. (a) Original image generated from section 2.3.1 of UPN251 cells. (b) Image ‘Drawing of X.JPG’ generated after analysis step 7, represents the cells counted from the analysis of original image. (c) Image ‘Drawing of X.JPG’ after accuracy step 5. This is the image that is used in the overlay onto the original image, to check for accuracy of the generated count versus the cells present in original image. (d) The result of the merged images (‘a’ and ‘c’) after completion of accuracy steps. In this case the count looks accurate as each cell is labelled with red numbering.
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fig0010: Example of images generated from the AF calculation method. (a) Original image generated from section 2.3.1 of UPN251 cells. (b) Image ‘Drawing of X.JPG’ generated after analysis step 7, represents the cells counted from the analysis of original image. (c) Image ‘Drawing of X.JPG’ after accuracy step 5. This is the image that is used in the overlay onto the original image, to check for accuracy of the generated count versus the cells present in original image. (d) The result of the merged images (‘a’ and ‘c’) after completion of accuracy steps. In this case the count looks accurate as each cell is labelled with red numbering.

Mentions: The microscope must be focused so that the cell nuclei are clearly visible on the camera’s LCD display. The cytoplasmic region of the cell will therefore be slightly out of focus. When using a different camera and microscope, the zoom settings may differ. In this case the camera must be zoomed in consistently so that cells appear as in Fig. 2(a) on the LCD display screen.


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)

Example of images generated from the AF calculation method. (a) Original image generated from section 2.3.1 of UPN251 cells. (b) Image ‘Drawing of X.JPG’ generated after analysis step 7, represents the cells counted from the analysis of original image. (c) Image ‘Drawing of X.JPG’ after accuracy step 5. This is the image that is used in the overlay onto the original image, to check for accuracy of the generated count versus the cells present in original image. (d) The result of the merged images (‘a’ and ‘c’) after completion of accuracy steps. In this case the count looks accurate as each cell is labelled with red numbering.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig0010: Example of images generated from the AF calculation method. (a) Original image generated from section 2.3.1 of UPN251 cells. (b) Image ‘Drawing of X.JPG’ generated after analysis step 7, represents the cells counted from the analysis of original image. (c) Image ‘Drawing of X.JPG’ after accuracy step 5. This is the image that is used in the overlay onto the original image, to check for accuracy of the generated count versus the cells present in original image. (d) The result of the merged images (‘a’ and ‘c’) after completion of accuracy steps. In this case the count looks accurate as each cell is labelled with red numbering.
Mentions: The microscope must be focused so that the cell nuclei are clearly visible on the camera’s LCD display. The cytoplasmic region of the cell will therefore be slightly out of focus. When using a different camera and microscope, the zoom settings may differ. In this case the camera must be zoomed in consistently so that cells appear as in Fig. 2(a) on the LCD display screen.

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