Quantitative Cell Attachment Assay

 

Objectives

·        Quantitatively assess attachment of HDF cells to different surfaces.

·        Assess variability among measurements.

 

Materials

24-well plates: TC-treated, untreated, Fibronectin (Fn)-coated

   NOTE:  The surface area of each well in a 24-well plate is 2 cm².

HDF cells

DMEM with 10% serum and 1% antibiotic

Tally counter

 

Safety

1.      Wear disposable latex gloves at all times

2.      Wear safety glasses at all times.

 

Test Conditions

Each person should evaluate two conditions, as listed below. 

 

Partner #1	Partner #2
TC-treated polystyrene	TC-treated polystyrene
untreated polystyrene	Fn-coated polystyrene

 

·        For each condition for each partner, 4 time points (30 min, 1 hr 15 min, 2 hr 30 min, 4 hr) should be tested.

·        For each time point, 3 wells should be run (i.e. each time point run in triplicate).

 

Prepare cells (accomplished by T.A. or Instructor)

1.      Proceed through cell passaging protocol until step #15. 

2.      After centrifugation, resuspend the cells in 10 mL DMEM with 10% serum. 

 

Procedure

NOTE:  Partners are to share a microscope.  Plan ahead and stagger the times for reading the attachment results!

1.      Lay out and label 24-well plates with different conditions. 

2.      Determine the cell concentration using the Coulter Counter.  Remember to correctly account for dilutions and Coulter Counter sampling volume.

3.      Determine the total volume of cell suspension that is to be seeded.  Remember, you have 2 plates, 4 time points, and each condition is run in triplicate.  You will need an additional 3 mL for step 7. Always prepare a little extra. 

4.      Dilute cell suspension (from stock) in DMEM with 10% serum and 1% antibiotic to a concentration of 10,000 cells/mL in 50 mL centrifuge tube.

5.      Seed 1 mL of cell suspension into each test well on your plates.

NOTE:  When seeding, mix the cells gently and often (every 6 wells or every 15 sec) to prevent settling of the cells.  It is VERY important to seed the same number of cells into each well for this assay!

6.      Place plates in incubator for 30 min.  Record the time the cells went into the incubator.

7.      While the plates are in the incubator, prepare 3 Coulter Counter vials, each with 9 mL Isoton.  Aliquot 1 mL of the remaining diluted cell suspension into each vial.  Determine the cell concentration using the Coulter Counter.

8.      After the allotted incubation time, remove the plates from the incubator. 

9.      Aspirate the media from the 3 wells for the appropriate time point on each plate.  Rinse wells with PBS using the following procedure:

a)      Add 1 mL of PBS to each well using a micropipette.

b)      Swish lightly.

c)      Aspirate PBS using glass Pasteur pipet.

d)      Repeat a) - c) two more times.

NOTE:  When adding PBS to each well, squirt the liquid on side wall of well (where there are no attached cells) rather than in center of well (where there are cells)!

NOTE:  Tilt plate slightly to assist in pooling liquid for aspiration. 

NOTE:  To streamline procedure, aspirate all wells, then add PBS to all wells, etc.

10.  Add 1 mL PBS to each rinsed well.  (i.e., leave PBS on cells for counting procedure).

11.  Count the number of attached cells in each rinsed well using a microscope.  Use the 10X objective.  Locate a place within each well where the cell density is representative of the whole area.  Count the number of cells in the 10x10 grid.  (Often the “wall” areas of a well are not representative, so do not consider this area.) 

NOTE:  When you are not actually counting cells on a plate, leave it in the incubator.

12.  Record the morphology, shape and “spreadness” of the cells.

13.  Return plates to incubator.

14.  Repeat Steps 8.-13. at 1 hr 15 min after the cells were seeded.

15.  Repeat Steps 8.-13. at 2 hr 30 min after the cells were seeded.

16.  Repeat Steps 8.-13. at 4 hr after the cells were seeded.

17.  Dispose of biohazardous materials properly.

 

Calculations/Questions

1.      (10 pts)  From the cell counts using the microscope, determine cell density in units of #-cells/area. When using a 10X objective (100X magnification), the area of the grid is 0.01 cm². 

2.      (10 pts)  Calculate the mean cell density and the standard deviation at the four measured time points for your two plates.

3.      (15 pts)  Share your data with your partner.  Record his/her data in your lab notebook.  Plot your data and that of your lab partner in a meaningful way.  Use calculated cell density rather than raw cell count data.

4.      (10 pts)  How does the rate of cell attachment vary among test conditions?  Comment on cell density and morphology.

NOTE:  Because of difference in technique, only compare trends within one partner’s data set in this and subsequent questions.

5.      (10 pts)  How does the “final” attachment density at 4 hrs vary among test conditions?  Include appropriate statistical analysis and comparisons.  Comment also on cell density and morphology.

6.      (10 pts)  Compare the measured cell density with a “theoretical” cell density (Step 7.). If they are dissimilar, hypothesize an explanation. Based on your data, is a 4 hr incubation long enough to ensure attachment before proceeding to another step in an experimental protocol?

7.      (15 pts)  How do your results from this experiment compare and contrast with your results from the “Fibronectin Attachment Assay”?

 

Answers to Calculations/Questions should be recorded in your laboratory notebook.  The point values are listed above.