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Week12 Adhesion

This document outlines a lab procedure focused on cell adhesion using various coatings on coverglasses and the role of integrins in cell attachment. The objectives include applying aseptic techniques and measuring cell adhesion on different surfaces. The procedure details steps for seeding cells, checking adhesion, and analyzing data, emphasizing the importance of recording observations and cleaning up after the experiment.

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12 views3 pages

Week12 Adhesion

This document outlines a lab procedure focused on cell adhesion using various coatings on coverglasses and the role of integrins in cell attachment. The objectives include applying aseptic techniques and measuring cell adhesion on different surfaces. The procedure details steps for seeding cells, checking adhesion, and analyzing data, emphasizing the importance of recording observations and cleaning up after the experiment.

Uploaded by

mgv1493
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Week 12 – Cell Adhesion

Introduction
Cells use integrins to attach to their surroundings including on surfaces in vitro. Integrins are
transmembrane proteins that adhere specifically to an amino acid sequence. Therefore, cells use
integrins to attach only to proteins. Coatings are surface modifications which can be used to
promote or inhibit cell adhesion, depending on the type of implant. Controlling this cell-surface
interaction is very important. Surfaces can be coated with proteins to enhance cell adhesion or
the proteins in the media with the cells may quickly adsorb to the surface to control cell
adhesion.

In this lab, coverglasses will be coated with substances: poly-L-lysine, bovine serum albumin
(BSA), fibronectin, polyethylene glycol (PEG). One coverglass will remain untreated as a
control. You will seed adhesion-dependent cells onto these five coverglasses and observe the
interactions between the cells and the differently coated surfaces using a microscope. It will be
important to record your observations. This is a procedure to determine which coatings promote
cell adhesion.

Objectives
 Apply aseptic technique for biomaterial testing.
 Measure adhesion of cells onto a variety of coated surfaces.

Prelab
A. Read lab procedure. Come to lab with any questions regarding the
procedure. Wear appropriate attire for a wet lab.
B. Write a hypothesis that you will either support or reject by the end of the
experiment.
C. Provide a literature reference supporting whether you expect fibroblasts or
any generic eukaryotic cell to adhere to each of the coatings. Print or submit
to Dropbox the abstract or other supporting material.
D. Make sure someone in your group has a USB drive for lab.

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Materials
70% ethanol in spray bottle Kimwipes
Pre-treated 12-well plate Flask of 3T3-L1 fibroblasts
Trypsin 5mLs Sterile PBS
16mLs Protein-Free Media Orbital shaker

Procedure: Seed Cells


A. Check the cells under a microscope. Write down observations about their
condition: confluency, shape, media color, and general appearance. This is
a basis for comparison.
a. You may take pictures but in order to include them in the report you must be able
to add a scale bar. The instructor can explain how that can be accomplished.
B. Lift cells from the flask.
a. Remove the media.
b. Rinse the cells with a few mLs of sterile PBS.
c. Remove the PBS.
d. Spread sterile trypsin over the cells.
e. Allow the cells 3-5 minutes to detach. Check their progress under a microscope.
f. Add 10mLs of protein-free media. Rinse the surface where the cells were
attached and pipet up and down to disrupt cell clumping.
C. Add cells to wells. To save time, you will evenly distribute the cell solution
between the samples without counting the cells.
a. Add 2mLs of the cell suspension to each of the 5 wells of your plate being used.
b. Allow the plate to incubate at 37°C for 25 minutes.
D. Put your group’s initials on your tube of protein-free media and place it back
in the water bath.

Procedure: Check Cell Adhesion


A. After the 25 minute incubation, place the plate on the orbital shaker.
B. Shake for 20 seconds at 500 rpm. Wait 10 seconds and repeat.
a. Turn on the shaker and let it get up to speed before placing your plate on it.
Leave it on between applications.
C. In the BSC, remove the media.
D. Add 1mL of fresh protein-free media to each of the wells being used.

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E. Perform a cell concentration determination.
a. Using the EVOS, determine if you are on top of or under the coverglass. This can
be accomplished by starting on the edge of the glass where there are cells on the
tissue-culture plastic. Find the focal plane where cells are in focus only on the
glass.
b. Using the 20x objective, identify a spot on the coverglass that represents the cell
density across the coverglass. Ideally choose a random spot.
c. Take a picture.
d. Obtain three pictures per sample.

Before you leave


 Turn in the carbon copies of you notebook.
 Add bleach to the flasks and plates and leave in containers next to sink.
 All used pipettes and tips get disposed of in the sharps box.
 Inform the instructor if the liquid waste flask in the BSC is turning pink.
 Clean your space.

Data Analysis

A. Discuss the observed differences in morphology (cell shape and spreading)


depending on how long you had to wait to take pictures.
B. In the “Equations” section, demonstrate how a count was achieved as well as
how the calculation was completed.
a. The field of view for the 20x objective on the EVOS independent of resolution is
0.88mm^2.
C. The “Final Analyzed Data” section display two-dimensional cell densities
with the coatings and what types of molecules they are.
a. The standard deviation is not calculated here because the counts are not from
independent samples.
b. Include a representative picture for each coating which includes the scale bar.

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