A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Knoll, Samantha; Ali, M. Yakut; Saif, M. Taher A.

doi:10.3791/51873

Cited by 39 publications

(41 citation statements)

References 18 publications

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“…Other approaches for depositing beads uniformly near the cell-gel interface have been published, including using a two-layer approach [50] or positively charging the top coverslip to coat it with beads [51]. While we have not compared these approaches side-by-side, they have predominantly been applied to single cell traction force microscopy of stiffer gels.…”

Section: Resultsmentioning

confidence: 99%

Monitoring developmental force distributions in reconstituted embryonic epithelia

Przybyla

Lakins

Sunyer

et al. 2016

Methods

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The way cells are organized within a tissue dictates how they sense and respond to extracellular signals, as cues are received and interpreted based on expression and organization of receptors, downstream signaling proteins, and transcription factors. Part of this microenvironmental context is the result of forces acting on the cell, including forces from other cells or from the cellular substrate or basement membrane. However, measuring forces exerted on and by cells is difficult, particularly in an in vivo context, and interpreting how forces affect downstream cellular processes poses an even greater challenge. Here, we present a simple method for monitoring and analyzing forces generated from cell collectives. We demonstrate the ability to generate traction force data from human embryonic stem cells grown in large organized epithelial sheets to determine the magnitude and organization of cell–ECM and cell–cell forces within a self-renewing colony. We show that this method can be used to measure forces in a dynamic hESC system and demonstrate the ability to map intracolony protein localization to force organization.

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Section: Resultsmentioning

confidence: 99%

Monitoring developmental force distributions in reconstituted embryonic epithelia

Przybyla

Lakins

Sunyer

et al. 2016

Methods

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“…Consequently, gels with relatively non-homogenous bead distribution can be discarded. Another approach is to use a recently proposed method that allows precise localization of the beads near the cell culture surface 17 .…”

Section: Discussionmentioning

confidence: 99%

“…Note: For visual demonstration of Section 3, the viewers/readers are referred to a recent Journal of Visualized Experiments (JoVE) article 17 .…”

Section: Preparation and Functionalization Of Different Stiffness Polmentioning

confidence: 99%

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Ali

Anand

Tangella

et al. 2015

JoVE

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Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

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“…Polyacrylamide (PAA) hydrogels with one-plane embedded fluorescent beads were prepared for traction force assays by following the method published by Knoll et al [21] with some modifications. Briefly, clean No.…”

Section: Methodsmentioning

confidence: 99%

The critical chemical and mechanical regulation of folic acid on neural engineering

et al. 2018

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The mandate of folic acid supplementation in grained products has reduced the occurrence of neural tube defects by one third in the U.S since its introduction by the Food and Drug Administration in 1998. However, the advantages and possible mechanisms of action of using folic acid for peripheral nerve engineering and neurological diseases still remain largely elusive. Herein, folic acid is described as an inexpensive and multifunctional niche component that modulates behaviors in different cells in the nervous system. The multiple benefits of modulation include: 1) generating chemotactic responses on glial cells, 2) inducing neurotrophin release, and 3) stimulating neuronal differentiation of a PC-12 cell system. For the first time, folic acid is also shown to enhance cellular force generation and global methylation in the PC-12 cells, thereby enabling both biomechanical and biochemical pathways to regulate neuron differentiation. These findings are evaluated in vivo for clinical translation. Our results suggest that folic acid-nerve guidance conduits may offer significant benefits as a low-cost, off-the-shelf product for reaching the functional recovery seen with autografts in large sciatic nerve defects. Consequently, folic acid holds great potential as a critical and convenient therapeutic intervention for neural engineering, regenerative medicine, medical prosthetics, and drug delivery.

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A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Cited by 39 publications

References 18 publications

Monitoring developmental force distributions in reconstituted embryonic epithelia

Monitoring developmental force distributions in reconstituted embryonic epithelia

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

The critical chemical and mechanical regulation of folic acid on neural engineering

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