Karyotyping of the B16-F10 Mouse Melanoma Cell Line Christine Widgren Advisor: Dr. Spilatro
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Chromosome Dropping The final cell suspension in 3:1 methanol:acetic acid fixative was then dropped from varying heights: 0 cm, 1 cm, and 30 cm. A ringstand holding a micropipetter was used to set the height of the cell suspension above the slide and the suspension could then be accurately dropped onto the slide from the appropriate height. 12 µl of cell suspension was dropped onto each slide and the slide was allowed to air dry completely. Staining Techniques Two different stains were used to visualize the chromosomes: DAPI fluorescent stain and Giemsa stain. Research shows that these stains produce similar banding patterns, so they should yield identical chromosome counts for the cell line. For slides stained with DAPI, the stain solution was placed onto the slide and then a cover slip was placed over the area, with the edges of the cover slip sealed using clear nail polish. For slides stained with Giemsa, the slide was stained for 30 seconds with Giemsa and then rinsed thoroughly with distilled water. The slide was then allowed to air dry. Slides were viewed under 100x oil immersion. Karyotyping of the B16-F10 Mouse Melanoma Cell Line Christine Widgren Advisor: Dr. Spilatro Cancer is prevalent within our society today, so as a means of better understanding complex mechanisms used by cancerous cell lines in humans, researchers are turning to animal models, such as the mouse model to simplify the complexity. Cancerous cell lines, such as the B16-F10 mouse melanoma cell line are categorized by chromosomal abnormalities, including missing/extra chromosomes (Buwe et al., 2003). In order to view the chromosomes of the cell line, a karyotype is produced, which is primarily used to show the number of chromosomes. Karyotypes can be obtained using a technique known as chromosome spreading, in which a cell suspension is dropped onto a slide to allow the cell contents to spread out and separate the chromosomes (Guttenbach et al., 2001). Once the spread is completed, different staining procedures can be employed to visualize the individual chromosomes (Guttenbach et al., 2001). Through visualization of chromosomes using karyotypes, the diploid number should be approxiatmely 2n = 54 (Stayon et al., 2004). However, this number may vary as rodents display the highest degree of chromosomal variation among all mammals (Stayon et al., 2004). The objective of this research was to determine whether the chromosome count from two different staining techniques, DAPI and Giemsa, would be identical, since previous research shows they have similar banding patterns. Cell Culturing B16-F10 mouse melanoma cells were cultured in CDMEM (DMEM, Penstrep, and FBS) for approximately three days before being transferred to new media. Two cell densities were kept: 50 µl and 100 µl. Cell Preparation In order to view the banding pattern of chromosomes, cells were arrested in metaphase using colcemid. After the cells were grown for 1-2 days, the cells were treated with a media containing colcemid (0.04 µg/mL) for six hours. Following the colcemid treatment, the cells were released from plates using trypsin and the cell suspension was then incubated at 37°C in a hypotonic solution of 0.075 M KCl for 30 minutes. The suspension was centrifuged and the supernatant removed. Cells were then resuspended in a 3:1 methanol:acetic acid fixative solution for 5 minutes. The solution was centrifuged and supernatant removed. Cells were washed four times using the fixative solution and in the final step were resuspended in 0.5 mL of From a qualitative perspective, cells dropped from 30 centimeters were much more spread out and had less overlapping of chromosomes than cells dropped from a height of one centimeter or zero centimeters. Using the metaphase spreads, the average chromosome count for the B16-F10 mouse melanoma cell line stained with DAPI was 2n=54 and the average chromosome count for slides stained with Giemsa was 2n=53. This number closely resembles the 54 chromosomes mice are said to have according to previous research. However, the chromosome count ranged from 2n=42 to 2n=64 on slides stained with DAPI and ranged from 2n=36 to 2n=62 on slides stained with Giemsa. This variability in chromosome count indicates the unstable nature of a cancerous cell line. I would like to thank Dr. Spilatro, who served as my capstone advisor for this research project, Dr. McShaffrey, who was our capstone class professor, and the Marietta College Biology Introduction Methods Results Literature Cited Acknowledgements Figure 1. Apparatus used for chromosome dropping. Figure 2. Sample metaphase spread showing intact and ruptured cells. Figure 3. DAPI stained metaphase spread. Cell suspension dropped from 30 cm. Figure 4. Giemsa stained metaphase spread. Cell suspension dropped from 1 cm. Deng W, Tsao SW, Lucas JN, Leung CS, Cheung AL. 2003. A new method for improving metaphase chromosome spreading. Cytometry Part A 51A: 46- 51. Guttenbach M, Steinlein C, Engel W, Schmid M. 2001. Cytogenetic characterization of the TM4 mouse Sertoli cell line. I. concentrional banding techniques, FISH and SKY. Cytogenetics and Cell Genetics 94: 71-78. Jentsch I, Adler ID, Carter NP, Speicher MR. 2001. Karyotyping mouse chromosomes by multiplex-FISH (M-FISH). Chromosome Research 9: 211-214. Rieder C, Palazzo R. 1992. Colcemid and the mitotic cycle. Journal of Cell Science 102: 387-392. Stanyon R, Yang F, Moreschalichi AM, Galleni L. 2004. Chromosome painting in the long-tailed field mouse provides insights into the ancestral murid karyotype. Cytogenetic and Genome Research 105: 406-411.
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Karyotyping of the B16-F10 Mouse Melanoma Cell Line Christine Widgren Advisor: Dr. Spilatro. Introduction. Results. Chromosome Dropping - PowerPoint PPT Presentation
Transcript of Karyotyping of the B16-F10 Mouse Melanoma Cell Line Christine Widgren Advisor: Dr. Spilatro
Chromosome Dropping The final cell suspension in 3:1 methanol:acetic acid fixative was then dropped from varying heights: 0 cm, 1 cm, and 30 cm. A ringstand holding a micropipetter was used to set the height of the cell suspension above the slide and the suspension could then be accurately dropped onto the slide from the appropriate height. 12 µl of cell suspension was dropped onto each slide and the slide was allowed to air dry completely.
Staining Techniques Two different stains were used to visualize the chromosomes: DAPI fluorescent stain and Giemsa stain. Research shows that these stains produce similar banding patterns, so they should yield identical chromosome counts for the cell line. For slides stained with DAPI, the stain solution was placed onto the slide and then a cover slip was placed over the area, with the edges of the cover slip sealed using clear nail polish. For slides stained with Giemsa, the slide was stained for 30 seconds with Giemsa and then rinsed thoroughly with distilled water. The slide was then allowed to air dry. Slides were viewed under 100x oil immersion.
Karyotyping of the B16-F10 Mouse Melanoma Cell Line
Christine WidgrenAdvisor: Dr. Spilatro
Cancer is prevalent within our society today, so as a means of better understanding complex mechanisms used by cancerous cell lines in humans, researchers are turning to animal models, such as the mouse model to simplify the complexity. Cancerous cell lines, such as the B16-F10 mouse melanoma cell line are categorized by chromosomal abnormalities, including missing/extra chromosomes (Buwe et al., 2003). In order to view the chromosomes of the cell line, a karyotype is produced, which is primarily used to show the number of chromosomes. Karyotypes can be obtained using a technique known as chromosome spreading, in which a cell suspension is dropped onto a slide to allow the cell contents to spread out and separate the chromosomes (Guttenbach et al., 2001). Once the spread is completed, different staining procedures can be employed to visualize the individual chromosomes (Guttenbach et al., 2001). Through visualization of chromosomes using karyotypes, the diploid number should be approxiatmely 2n = 54 (Stayon et al., 2004). However, this number may vary as rodents display the highest degree of chromosomal variation among all mammals (Stayon et al., 2004). The objective of this research was to determine whether the chromosome count from two different staining techniques, DAPI and Giemsa, would be identical, since previous research shows they have similar banding patterns.
Cell Culturing B16-F10 mouse melanoma cells were cultured in CDMEM (DMEM, Penstrep, and FBS) for approximately three days before being transferred to new media. Two cell densities were kept: 50 µl and 100 µl.
Cell Preparation In order to view the banding pattern of chromosomes, cells were arrested in metaphase using colcemid. After the cells were grown for 1-2 days, the cells were treated with a media containing colcemid (0.04 µg/mL) for six hours. Following the colcemid treatment, the cells were released from plates using trypsin and the cell suspension was then incubated at 37°C in a hypotonic solution of 0.075 M KCl for 30 minutes. The suspension was centrifuged and the supernatant removed. Cells were then resuspended in a 3:1 methanol:acetic acid fixative solution for 5 minutes. The solution was centrifuged and supernatant removed. Cells were washed four times using the fixative solution and in the final step were resuspended in 0.5 mL of fixative. (Procedures taken from Deng et al.)
From a qualitative perspective, cells dropped from 30 centimeters were much more spread out and had less overlapping of chromosomes than cells dropped from a height of one centimeter or zero centimeters. Using the metaphase spreads, the average chromosome count for the B16-F10 mouse melanoma cell line stained with DAPI was 2n=54 and the average chromosome count for slides stained with Giemsa was 2n=53. This number closely resembles the 54 chromosomes mice are said to have according to previous research. However, the chromosome count ranged from 2n=42 to 2n=64 on slides stained with DAPI and ranged from 2n=36 to 2n=62 on slides stained with Giemsa. This variability in chromosome count indicates the unstable nature of a cancerous cell line.
I would like to thank Dr. Spilatro, who served as my capstone advisor for this research project, Dr. McShaffrey, who was our capstone class professor, and the Marietta College Biology Department for funding this project.
Introduction
Methods
Results
Literature Cited
Acknowledgements
Figure 1. Apparatus used for chromosome dropping.
Figure 2. Sample metaphase spread showing intact and ruptured cells.
Figure 3. DAPI stained metaphase spread. Cell suspension dropped from 30 cm.
Figure 4. Giemsa stained metaphase spread. Cell suspension dropped from 1 cm.
Deng W, Tsao SW, Lucas JN, Leung CS, Cheung AL. 2003. A new method for improving metaphase chromosome spreading. Cytometry Part A 51A: 46-51.
Guttenbach M, Steinlein C, Engel W, Schmid M. 2001. Cytogenetic characterization of the TM4 mouse Sertoli cell line. I. concentrional banding techniques, FISH and SKY. Cytogenetics and Cell Genetics 94: 71-78.
Jentsch I, Adler ID, Carter NP, Speicher MR. 2001. Karyotyping mouse chromosomes by multiplex-FISH (M-FISH). Chromosome Research 9: 211-214.
Rieder C, Palazzo R. 1992. Colcemid and the mitotic cycle. Journal of Cell Science 102: 387-392.
Stanyon R, Yang F, Moreschalichi AM, Galleni L. 2004. Chromosome painting in the long-tailed field mouse provides insights into the ancestral murid karyotype. Cytogenetic and Genome Research 105: 406-411.
