Tissue Culture Laboratory (BIOE342) - Protocol
Index



Anti-PCNA Staining

 

Objectives

·        Observe connection between cell cycle/proliferation and media conditions.

·        Become familiar with antibody-specific staining procedures.

 

Materials

Antibody to Proliferating Cell Nuclear Antigen (Anti-PCNA) with Horseradish

   Peroxidase (HRP)

AEC chromogenic substrate solution

Negative control solution (HRP/FBS)

Formalin, 10% buffered

PBS

Methanol

3% Hydrogen Peroxide (H2O2) in DI water

Tris Buffer Saline (TBS)

37mM Ammonium Hydroxide (NH4OH)

Mayer’s hematoxylin, syringe filtered

DI water

24-well TC-treated plates - use plates from Live/Dead assay

Timer

HDF cells

DMEM with 1% serum and 1% antibiotic

DMEM with 5% serum and 1% antibiotic

DMEM with 10% serum and 1% antibiotic

 

Safety

1.      Wear disposable latex gloves at all times

2.      Wear safety glasses at all times.

3.      Formalin is toxin (may cause cancer).  It is absorbed through skin and inhalation (lungs).  Do not breathe vapors. 

4.      Methanol is flammable.  Do not place near open flame or extreme heat.

5.      H2O2 is an oxidizer and is corrosive.  Keep away from combustible materials.  Wear butyl, neoprene or vinyl gloves for additional protection.

 

Solution Preparation (by T.A. or Instructor)

1.      DMEM with 1%, 5%, and 10% FBS and antibiotic.

2.      37mM NH4OH:  2.5 mL of 15 M NH4OH in 1L DI water.  Prepare new each year.

3.      Tris Buffer Saline (TBS):  0.05 M Tris-HCl (Sigma), 0.15 mM NaCl, pH 7.6.

4.      3% H2O2: 1:10 dilution of 30% H2O2 in DI water.

 

Cell Preparation (Day 1)

NOTE:  Use 24-well plates from Live/Dead assay.  Each partner should be using his/her own plate. 

1.      Acquire cells from one of the following sources:

·        Cells harvested for Quantitative Cell Attachment Assay.

·        Confluent flask you are maintaining.

2.      Proceed through appropriate steps so that cells can be centrifuged.

3.      Centrifuge cells.  After centrifugation, aspirate supernatant.  Cell pellet should remain at base of tube.

4.      Resuspend cells in 10 mL of DMEM with 1% serum and 1% antibiotic.

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

6.      Prepare 10 mL of cell suspension at 20,000 cells/mL in each of the following:

·        DMEM with 1% serum and 1% antibiotic

·        DMEM with 5% serum and 1% antibiotic

·        DMEM with 10% serum and 1% antibiotic

NOTE:  Do not be concerned about the slight media dilution effect in this step of the protocol.

7.      Seed 1 mL of each prepared cell suspension with different serum concentrations into your 24-well plate. 

NOTE:  Because of the expense of the PCNA antibody, only one test at each condition will be conducted.

8.      Seed 2 additional wells with 1 mL of cells in DMEM with 10% serum and 1% antibiotic.  These two wells will serve as controls.

9.      Place cells in incubator for 2 days. 

 

PCNA Assay (Day 3)

NOTE:  All work can be done on bench (i.e. sterile conditions are NOT required). 

steps. 

NOTE:  Work gently with attached cells, as they tend to shear under repeated rinse conditions.  When adding liquid to each well, squirt the liquid on side wall of well rather than in center of well.

 

Waste

·        Screw-cap bottle for methanol rinses

·        Screw-cap bottle for formalin rinses

·        Screw-cap bottle for H2O2 rinses

·        Beaker for remainder of liquid waste

Since all wastes contacted cells, they should be considered biohazardous.

 

“Rinse” steps below include the following steps:

·        Add 1 mL liquid.

·        Swish around gently.

·        Aspirate using micropipette.

 

NOTE:  For steps 1. - 15., a micropipette can be used to conduct the rinse and aspiration steps.

1.      Check cells under microscope.  Record confluency and morphology.

2.      Aspirate media from well.

3.      Rinse cells gently with 1 mL PBS.

4.      Add 0.5 mL formalin to cells.  Let stand for 10 min.  Aspirate formalin.

5.      Rinse cells with 1 mL PBS two times.

6.      Add 0.25 mL methanol to cells.  Let stand for 2 min.  Aspirate methanol.

7.      Add 1 mL PBS to cells.  Let stand for 5 min.  Aspirate PBS.

8.      Add 1 mL 3% H2O2 to cells.  Let stand for 5 min.  Aspirate H2O2.

9.      Rinse cells with 1 mL DI water two times.

10.  Add 1 mL TBS to cells.  Let stand for 5 min.  Aspirate TBS.

11.  Add solution as described:

Ø      Depending on amount of available Anti-PCNA/HRP and the level of confluency of your test wells, only two of the three test conditions may have Anti-PCNA/HRP added.  Consult Dr. Saterbak.

·        For test conditions (1%, 5%, 10% serum) , add 125-150 mL of Anti-PCNA/HRP. 

·        For control #1, add 150 mL of negative control solution. 

·        For control #2, add 150 mL of TBS. 

Ø      Make sure that the antibody solution or control solution completely wets (i.e. covers) the well plate. 

12.  Let stand at room temperature for at least 60 min.

13.  Aspirate antibody and/or control solutions.

14.  Add 1 mL TBS to cells.  Let stand for 5 min.  Aspirate TBS.

 

NOTE:  For steps 15. - 20., a glass Pasteur pipet and the vacuum system in a laminar flow hood must be used for each aspiration step.  Do not worry about decontaminating the well plate each time it enters the hood;  however, be very careful to not contaminate other items such as pipet tip boxes, etc.  Only the aspiration step should be done in the hood.

15.  Add 2-3 drops of AEC solution (straight from fridge) to cells.  Let stand for 10 min.  (Do not exceed 10 min.)  Aspirate AEC solution.

16.  Rinse cells with 1 mL DI water 4-5 times. 

17.  Add 2-3 drops of hematoxylin to cells.  Let stand for 5 min. (Do not exceed 5 min.)  Aspirate hematoxylin.

18.  Rinse cells with 1 mL DI water.

19.  Add 1 mL 37mM NH4OH to cells.  Let stand for 1 min.  (Solution and/or cells should turn clear blue.)  Aspirate NH4OH.

20.  Rinse cells with 1 mL DI water 4-5 times. 

21.  View cells using light microscope. Remove green filter.  Focus on cells in center of well;  do not focus on cells at the perimeter of well.

22.  (20 pts)  Record observations.  Include colors of cell cytoplasms and nuclei.  Dividing cell nuclei should stain red.  Hematoxylin stains cell nuclei blue.

23.  View the results of lab partner or another student in the lab.

24.  Dispose of biohazardous and chemical wastes properly.

 

Questions

1.      (20 pts)  Are there differences among cells that were incubated at different conditions?  If so, what are they and why do you think that there are differences?

 

Observations and answers to Questions should be recorded in your laboratory notebook.  The point values are listed above.