Cell tracing dyes significantly change single cell mechanics

Abstract

Cell tracing dyes are very frequently utilized in cellular biology research because they provide highly sensitive fluorescent tags that do not compromise cellular functions such as growth and proliferation. In many investigations concerning cellular adhesion and mechanics, fluorescent dyes have been employed with the assumption of little impact on the results. Using the single cell compression technique developed by our team, the single cell mechanics of MDA-MB-468 and MLC-SV40 cells were investigated as a function of dye uptake. Cell tracing dyes increase living cell stiffness 3-6 times and cell-to-probe adhesion up to 7 times. These results suggest a more significant effect than toxins, such as thrombin. A simple analytical model was derived to enable the extraction of the Young's moduli of the cell membrane and cytoskeleton from the force-deformation profiles measured for individual cells. The increase in Young's modulus of the membrane is 3-7 times, which is more significant than that of the cytoskeleton (1.1-3.4 times). We propose that changes in cell mechanics upon the addition of fluorescent tracing dye are primarily due to the incorporation of amphiphilic dye molecules into the cellular plasma membrane, which increases the lateral interaction among phospholipid chains and thus enhances their rigidity and adhesion.

Figure 1

(A) A schematic diagram of the single cell compression experiment. (B) Cell deformation under pressure such as cytoskeleton compression and blebbing. Red meshes represent cytoskeleton. Red arrows illustrate the concept of transforming external force into the intracellular pressure.

Figure 2

Typical profiles, i.e. force versus relative deformation, for the investigation of CMFDA dye effect on MDA-MB-468. Curve 1 (red) represents untreated cell, curve 2 (gray) is DMSO treated cell. Curves 3 (blue) and curve 4 (color?) are deformation profiles for cell labeled with 3 and 5 μM CMFDA, respectively. Chemical structure of CMFDA is shown on right. The insert shows the loading portion of the profile at low deformations. Bottom row includes epifluorescent snapshots of 5 μM CMFDA labeled MDA-MB-468 cell taken during compression cycle (bottom view), from which blebs are clearly visible.

Figure 3

Typical force versus relative deformation profiles for MDA-MB-468 cells labeled with various dyes: a control cell (1, red), a EGTA treated cell (2, gray), and cells labeled with 5 μM CFDA-SE (3, blue), CMFDA (4, green), CMTMR (5, orange) and Calcein Green (6, black), respectively. The chemical structure of Calcein Green, EGTA and CFDA-SE are shown on the right.

Figure 4

Typical deformation profiles for the investigation of cell tracing dyes on MLC-SV40 cells: an unlabeled (control) cell (1, red), a DMSO treated cell (2, gray), and a cell labeled with 5 μM CFDA-SE (3, blue). The insert shows the loading portion of the profile at low deformations. Bottom row shows epifluorescent snapshots of a 5 μM CFDA-SE labeled MLC-SV40 cell taken during compression cycle (bottom view).

Figure 5

Schematic diagram of how CMFDA cell tracing dye molecules can incooperate into phospholipids bilayer increasing its rigidity. Polar regions are shown in orange, nonpolar in gray, cytoplasm is represented by red mesh.