Natalie Raso - Weapons of Targeted Destruction: Using Viruses to Kill Cancer
     Procedure for Cell-Line Splitting
Project Information

Abstract

Project Summary

Background

Purpose

Scientific Thought

Hypotheses

Apparatus and Materials

Genetically Engineered KM110red Herpesvirus

Methodology

Procedure for Cell-Line Splitting

Procedure for KM110r Infection

Procedure for Immunofluorescent
Microscopy Imaging

Statistical Analyses

Proliferation Assay Analyzed Data

Major Results

Graphed Results

Discussion of Statistics

Controls and Variables

Conclusions

Discussion

Discussion of KM110r Efficacy

Successes and Failures

Sources of Error and Data

Limitations

Future Research

Applications

Glossary

Bibliography

Acknowledgements 		 

In order for the cell lines (hFOB and U2OS) to continue growing at healthy levels, they require being split on a regular basis, usually 2 or 3 days. Depending on the level of cell confluence within the T75, the cells were split at a ratio. If the cells were less confluent they would be split 1:2 (meaning half of the cells would be maintained), if they were more confluent they could be split as high as 1:10 (meaning a tenth of the cells would be maintained). The steps for performing this procedure are outlined below. This procedure was carried out in a Level II Biohazard Tissue Culture Laboratory in a Level II Biosafety Cabinet.

 

  1. Determine the ratio of splitting that needs to be performed. (Example: 1:4)
  2. Remove media from T75 container with aspirating pipette.
  3. Rinse the T75 with 10mL PBS. Rock 3 or 4 times, then aspirate off.
  4. Add ½ mL Trypsin.
  5. Allow cells to trypsinize until cells release themselves from the inside of the T75.
  6. If the cells require 1:4 splitting, add 3½ mL media since there is still ½ mL trypsin. There would now be 4mL of solution in the T75.
  7. Draw up and release media 5 or 6 times in order to mix solution thoroughly.
  8. Draw up 1mL (1/4) of solution and add to new T75.
  9. Add 9ml of DMEM +10% FBS to new T75 and rock.
  10. Replace the new labelled T75 in incubator.

Once the hFOB and U20S cells were ready to be analyzed they were split from the T75 into 6-well dishes. The dishes contained two wells each of hFOB, U2OS, and MIX co-culture (50% U2OS, 50% hFOB) seeded at concentrations of 1.3x105 cells/mL. The steps for performing this procedure are outlined below. This procedure was carried out in a Level II Biohazard Tissue Culture Laboratory in a Level II Biohazard Cell Hood.

 

1.      Remove DMEM from each T75 with hose.

2.      Rinse each T75 with 10mL of PBS)

3.      Add ½ ml Trypsin to each T75.

4.      Allow cells to trypsinize until the cells release themselves from the inside of the T75.

5.      Determine the number of cells per mL in both the hFOB and U20S preparations using a haemocytometer.

6.      Resuspend both hFOB and U-20S cells at 1.5x105 cells per well.

7.      Divide cell concentration (see step 6) per mL by the number of cells x105 per mL, and multiply the quotient by 1000µl to determine the amount of cells to be deposited into each well.

8.      The volume of each well should be 2mL. Therefore the amount of DMEM for each well is determined by subtracting the amount of cells to be deposited into each well in µL, from 2mL.

9.      2mL of the hFOB mixture will be deposited into two of the 6-wells, as will 2mL of the U20S mixture.

10. Two of the wells will be ‘MIX’ wells, in which 1mL of the hFOB mixture and 1mL of the U20S mixture will be combined in the same well.

 



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