Read the protocol to a cell adhesion assay experiment:Introduction- While there are many approaches that can be taken to study the "Hallmarks of Cancer", some of the most economical assays can be done using tissue culture models. While we will soon embark on a discussion of the perils of relving solely on cells grown in culture, we will find that the benefits of their usage comes in their "tractability": agonists and antagonists can be readily applied and removed from the cellular environment and behavioral reactions can be measured in a matter of hours or days. Thus, by using models such as tissue culture cells scientists can pose "bite-sized" experimental hypotheses that can be tested in well-controlled experiments in a relatively short amount of time.We will take advantage of these benefits this week by performing a simple assay of adhesive behavior that is commonly performed in the Cancer Biology world to ask basic questions about tumor cell behavior. Today we will visualize cells as they exhibit various stages of adhesion. We will monitor the structures of the cells with a stain that detects the presence of filamentous actin (F-actin) as well as cell nuclei. The details of the experiment is described below.GOAL: By the completion of this lab exercise, you should be able to understand the connection between this basic assay of cell adhesion and those "Hallmarks" pertaining to migration, CM and tumor microenvironment.Cell Adhesion AssayExperiment: Each lab pair will receive a plate with two types of glass coverslips: plain or ECM-coated. Using jewler's forceps, transfer coverslips to the bottom of the wells of a 24 well plate as illustrated below (Note- on the cartoon in picture, gray circles indicate empty wells.)After coverslips have been placed in the wells, cover each one with 0.5 mL media.Prepare suspensions of Fibroblast cells by trypsinizing adherent cells off of their stock flasks:• Aspirate media using the vacuum flask apparatus•Wash monolayer of cells with 1 mL. warm trypsin• Aspirate trypsin•Add 0.5 mL fresh trypsin solution• Incubate at 37 degrees for 30 seconds• Slap flask and look through bottom of the flask towards thelight to make sure cells are being dislodged from the plastic•Add 10 mL of media to the flask to inhibit the trypsin• The cells will be in a suspension of ~ 50,000 cells per mL.Seeding wells:•At the appropriate time point, aspirate media off of the coverslip• Take the tube containing the stock Fibroblast cellsuspension and add 1.0 mL of the suspension to the appropriate wells. (Note - it is probably best to add cells to the longest time point first and then work backwards through t=0 minutes - that way all the coverslips can be harvested/fixed/stained at the same time.•Working quickly, return the plate to the humidified 37°Cincubator in the tissue culture room.To terminate the adhesion assay•When all of the coverslips have been seeded. Each coverslip should be washed 2 times with 1 mL 1x PBS, aspirating/pipetting the liquid between each wash.After the last of the second PBS wash is removed, add 0.5 mL of PHEMO-Fix to each well/coverslip and incubate at room temperature for 10 minutes. • To stop the fixative reaction, wash each well 2x more with 1X PBS as above – 5 minutes each wash on shaker. • After the fixative is successfully removed and inactivated, apply Staining Solution (green dye for F-Actin, blue dye for nuclei) to each coverslip for 20-30 minutes (cover with aluminum foil). • At the end of that incubation wash each coverslip twice in 1X PBS and 1X with 1 mL ddH2O. • Mount on labeled slides – no more than 2 coverslips per slide, using Anti-Fade mounting media1. Based on your figure of the cells under the microscope in the picture (10x magnification) , list the 3 most compelling results of your experiment (i.e. 3 results, NOT conclusions)2. Explain why you think each of these results is important (i.e., how do these results support/fail to support the experimental prediction and the hypothesis.3. If you had to write a one sentence title describing your figure’s “bottom line”, what would it be? -ECM+ECMT=0 T=10 T=30 T=60OOOT=1205000OOOOOOOOOOGOOOOCoverslip# 1-5#6-10 DAPI: ECMDAPI: NoECMT=0T=30T=60T=120

Question

Read the protocol to a cell adhesion assay experiment:Introduction- While there are many approaches that can be taken to study the &#34;Hallmarks of Cancer&#34;, some of the most economical assays can be done using tissue culture models. While we will soon embark on a discussion of the perils of relving solely on cells grown in culture, we will find that the benefits of their usage comes in their &#34;tractability&#34;: agonists and antagonists can be readily applied and removed from the cellular environment and behavioral reactions can be measured in a matter of hours or days. Thus, by using models such as tissue culture cells scientists can pose &#34;bite-sized&#34; experimental hypotheses that can be tested in well-controlled experiments in a relatively short amount of time.We will take advantage of these benefits this week by performing a simple assay of adhesive behavior that is commonly performed in the Cancer Biology world to ask basic questions about tumor cell behavior. Today we will visualize cells as they exhibit various stages of adhesion. We will monitor the structures of the cells with a stain that detects the presence of filamentous actin (F-actin) as well as cell nuclei. The details of the experiment is described below.GOAL: By the completion of this lab exercise, you should be able to understand the connection between this basic assay of cell adhesion and those &#34;Hallmarks&#34; pertaining to migration, CM and tumor microenvironment.Cell Adhesion AssayExperiment: Each lab pair will receive a plate with two types of glass coverslips: plain or ECM-coated. Using jewler's forceps, transfer coverslips to the bottom of the wells of a 24 well plate as illustrated below (Note- on the cartoon in picture, gray circles indicate empty wells.)After coverslips have been placed in the wells, cover each one with 0.5 mL media.Prepare suspensions of Fibroblast cells by trypsinizing adherent cells off of their stock flasks:• Aspirate media using the vacuum flask apparatus•Wash monolayer of cells with 1 mL. warm trypsin• Aspirate trypsin•Add 0.5 mL fresh trypsin solution• Incubate at 37 degrees for 30 seconds• Slap flask and look through bottom of the flask towards thelight to make sure cells are being dislodged from the plastic•Add 10 mL of media to the flask to inhibit the trypsin• The cells will be in a suspension of ~ 50,000 cells per mL.Seeding wells:•At the appropriate time point, aspirate media off of the coverslip• Take the tube containing the stock Fibroblast cellsuspension and add 1.0 mL of the suspension to the appropriate wells. (Note - it is probably best to add cells to the longest time point first and then work backwards through t=0 minutes - that way all the coverslips can be harvested/fixed/stained at the same time.•Working quickly, return the plate to the humidified 37°Cincubator in the tissue culture room.To terminate the adhesion assay•When all of the coverslips have been seeded. Each coverslip should be washed 2 times with 1 mL 1x PBS, aspirating/pipetting the liquid between each wash.After the last of the second PBS wash is removed, add 0.5 mL of PHEMO-Fix to each well/coverslip and incubate at room temperature for 10 minutes.  • To stop the fixative reaction, wash each well 2x more with 1X PBS as above – 5 minutes each wash on shaker. • After the fixative is successfully removed and inactivated, apply Staining Solution (green dye for F-Actin, blue dye for nuclei) to each coverslip for 20-30 minutes (cover with aluminum foil).   • At the end of that incubation wash each coverslip twice in 1X PBS and 1X with 1 mL ddH2O.   • Mount on labeled slides – no more than 2 coverslips per slide, using Anti-Fade mounting media1. Based on your figure of the cells under the microscope in the picture (10x magnification) , list the 3 most compelling results of your experiment (i.e. 3 results, NOT conclusions)2. Explain why you think each of these results is important (i.e., how do these results support/fail to support the experimental prediction and the hypothesis.3. If you had to write a one sentence title describing your figure’s “bottom line”, what would it be? -ECM+ECMT=0 T=10 T=30 T=60OOOT=1205000OOOOOOOOOOGOOOOCoverslip# 1-5#6-10 DAPI: ECMDAPI: NoECMT=0T=30T=60T=120

Accepted Answer

The extracellular matrix (ECM) is an intricate network consisting of extracellular macromolecules…