Plant and Mammalian Tissue Culture

1. Plant and Mammalian Tissue Culture Counting Cells and Experimental Design

2. Counting Cells Estimating the number of cells is important in maintaining and freezing cells lines. You should have the laboratory prepared to do it quickly and easily.

3. A device originally designed for the counting of blood cells. It is now also used to count other types of cells as well as other microscopic particles Hemocytometer

4. The ruled consists of several, large, 1 x 1 mm (1 mm2) squares. These are subdivided in 3 ways: 0.25 x 0.25 mm (0.0625 mm2) 0.25 x 0.20 mm (0.05 mm2) 0.20 x 0.20 mm (0.04 mm2) The central, 0.20 x 0.20 mm marked, 1 x 1 mm square is further subdivided into 0.05 x 0.05 mm (0.0025 mm2) squares Grid Lines

5. The raised edges of the hemocytometer hold the coverslip 0.1 mm off the marked grid. This gives each square a defined volume. Coverslip

6. Volume

7. Accurate cells counts require a uniform cell suspension and appropriate loading of the hemocytometer Loading

8. The 20 ml pipette tip is placed on the hemocytometer at the edge of the coverslip. The plunger of the adjustable pipette is slowly pressed so that the sample flows between the hemocytometer's raised shiny surface and the coverslip. Stop when the sample touches the three sides of the chamber (about 10 ml) Do not overflow into the moat which surrounds the chambers. Loading

9. When counting mammalian cells, using the 9 large 1mm x 1 mm squares is adequate. Counting

10. Keep track of the number of cells counted and the number of 1mm2 squared counted. Counting

11. Use a consistent method for counting cells in the lines. Example Count cells on top line and right side Do not count left line and bottom This keeps a researcher from double counting or missing cells Counting

12. Cells / ml • To calculate the cells/ml when counting 1mm2 areas. • Total # cells x 10,000 = cells / ml# of Squares • A 1 mm2 area that is 0.1 mm deep is a volume of 100 nl • 100 nl x 10,000 = 1 ml

13. A vital stain used to selectively color dead tissues or cells blue Trypan Blue

14. Method • Prepare a mixture of cell suspension and a 0.4% trypan blue solution. • An equal volume dilution is common • 25 µl cell suspension and 25 µl 0.4% trypan blue

15. Count living cells White cells Count dead cells Blue cells % Viable Cells = Living Cells x 100Total Cells Viability

16. Remember that if you use a viablilty count to also determine cells / ml you need to include the dilution factor. 50% of volume was trypan blue solution Must multiply cell counted x2 to get cells /ml in original suspension Viability

17. Subculture and Growth Curve Part I Student groups start culturing cells in T-25 flasks This is a simple culture exercise. The purpose is to become familiar with cell culturing techniques. Your laboratory notebook will have the times you cultured, purpose and outline of the procedures you used. Do NOT paste protocols into the lab book. Instead, reference the protocols and write an outline/flow chart / bulleted list of what you did. Include notes on the following: • Cell culture used • Confluence & morphology of the cells • Color & appearance of media • Calculations for culturing • Calculations and cell counts

18. Subculture and Growth Curve Part I Student groups start culturing cells in T-25 flasks This is a simple culture exercise. The purpose is to become familiar with cell culturing techniques. Experimental Procedure: • Culture 1:4 splits (keep one flask each time) • Split cells every other day for four total subcultures. • Count cells from old flask AND calculate number of cells cultured into new flask EACH time. • Work with partner over weekend (OR split cells higher on Friday and Culture again on Monday – check with instructor before doing this). • Ensure you have two flasks prepared on Sunday for Monday!

19. Subculture and Growth Curve Part II Growth Curve Determination (Monday). In this exercise, each group will seed several 6-well dishes with an identical number of cells. Then at the same time each day (+/- 3 hours) one person from the group will trypsinize the cells and count three of the wells for that day. In the notebook ensure the following is recorded: • Cell culture used • Confluence & morphology of the cells • Color & appearance of media • Calculations and cell counts • A final graph (with error bars) of the growth curve. Indicate the each phase of the cell growth. • Use the ATCC or NCBI library to determine where the cells came from and a simple description of your cells.

20. Subculture and Growth Curve Part II Growth Curve Determination (Monday). In this exercise, each group will seed several 6-well dishes with an identical number of cells. Experimental Procedure: • Using the two flasks from your T-25 culturing, seed three 6-well dishes with 100,000 cells per each well. Two are for counting one is just in case a mistake is made! • Each day (Tuesday – Friday) one person from the group will trypsinize 3 wells and count the cells. Record the conditions as listed above each time and share with the group. • By Friday, each group should have four days of cell counts. IF the cells reach 100% confluence, continue as directed above, but carefully watch the conditions of the cells. They may change morphology or start to die off. This is also important data. • Prepare the graph as indicated above.