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. 2014 Jan;9(1):182-92.
doi: 10.1038/nprot.2014.004. Epub 2014 Jan 2.

Determining the macropinocytic index of cells through a quantitative image-based assay

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Determining the macropinocytic index of cells through a quantitative image-based assay

Cosimo Commisso et al. Nat Protoc. 2014 Jan.

Abstract

Macropinocytosis serves as an internalization pathway for extracellular fluid and its contents. Macropinocytosis is upregulated in oncogene-expressing cells and, recently, we have revealed a functional role for macropinocytosis in fueling cancer cell growth through the internalization of extracellular albumin, which is degraded into a usable source of intracellular amino acids. Assessing macropinocytosis has been challenging in the past because of the lack of reliable assays capable of quantitatively measuring this uptake mechanism. Here we describe a protocol for visualizing and quantifying the extent of macropinocytosis in cells both in culture and growing in vivo as tumor xenografts. By using this approach, the 'macropinocytic index' of a particular cell line or subcutaneous tumor can be ascertained within 1-2 d. The protocol can be carried out with multiple samples in parallel and can be easily adapted for a variety of cell types and xenograft or allograft mouse models.

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Conflict of interest statement

The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1. Representative images typically obtained from an in vitro macropinocytosis assay
The cells utilized in this particular assay were MIA PaCa-2 (top panel) and BxPC-3 (bottom panel). Nuclei are labeled with DAPI. A phase contrast image is used to determine the area of the field covered by cells. Fluorescent images captured in the rhodamine channel show the TMR-dextran-positive macropinocytic puncta for each field. For display purposes only, the TMR-dextran images were adjusted for brightness and contrast via ImageJ by selecting the B&C auto function. This adjustment does not affect the subsequent thresholding of the images. To determine the threshold value to be applied to all the images, the MIA PaCa-2 image was utilized. Based on this image, a threshold of 133 (on a dark background) was applied to all the images. Prior to accepting the threshold value, ImageJ depicts the detected macropinosomes in red (Set Threshold). Once accepted, the image is converted to a binary image in which the macropinosomes are shown in black on a white background (Binary Image). In the binary image, only particles that fall within the cell-covered area (yellow outline) contribute to the calculation of the ‘Field Index’. Scale bar represents 20 μm.
Figure 2
Figure 2. Representative images typically obtained from an in vivo macropinocytosis assay
Tumors used in this assay were derived from MIA PaCa-2 (top panel) or BxPC-3 (bottom panel) cells. Nuclei are labeled with DAPI. The pancreatic epithelial cells are labeled with the Troma I antibody, which labels Cytokeratin-8 (CK-8) and areas of the tumor populated by the CK-8-positive epithelial cells are outlined in yellow. Fluorescent images captured in the FITC channel show the FITC-dextran-positive macropinocytic puncta for each field. The CK-8 and FITC-dextran images were automatically adjusted for brightness and contrast via ImageJ. The automated threshold value was determined for each individual tumor (MIA PaCa-2 = 378, BxPC-3 = 464) and verified visually. When setting the threshold, ImageJ depicts the macropinosomes that fall within the threshold setting as red (Set Threshold) and converts the image to a binary image for quantification (Binary Image). In the binary image, only particles that fall within the CK-8-positive area (yellow outline) contribute to the computation of the ‘Field Index’. Appropriate institutional regulatory board permission was obtained for the animal experiments this data was derived from. Scale bar represents 20 μm.

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