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* Remove medium, add fresh 0.1% trypsin for about 10 minutes, and centrifuge to remove trypsin. Resuspend cell pellet in fresh medium and dispense into new flasks. Subculture every 7 days.
* Remove medium, add fresh 0.1% trypsin for about 10 minutes, and centrifuge to remove trypsin. Resuspend cell pellet in fresh medium and dispense into new flasks. Subculture every 7 days.
===Peer assessment comments===

Revision as of 10:11, 12 May 2011

Group 6

Neuromuscular Junctions

User:z3219008 | User:z3255968 | User:z3347321 | User:z3265772



Lab 1. --z3265772 10:35, 10 March 2011 (EST)

Lab 2. --z3265772 09:10, 17 March 2011 (EST)

Lab 3. --z3265772 09:37, 24 March 2011 (EST)

Lab 4. --z3265772 09:22, 31 March 2011 (EST)

Lab 5. --z3265772 09:17, 7 April 2011 (EST)

Lab 6. --z3265772 11:03, 14 April 2011 (EST)

Lab 7. --z3265772 08:48, 21 April 2011 (EST)

Lab 8. --z3265772 11:12, 5 May 2011 (EST)

Lab 9. --z3265772 09:42, 12 May 2011 (EST)

Lab 10.

Lab 11.

Lab 12.

Lab Questions

Lab 1 Questions

1. What are the key cell biology journals?

The journal of cell biology, Cell, Nature cell biology, BMC cell biology, Molecular and cell biology

2.Which journals allow reuse of their published content?

The journal of cell biology (when cited), Cell (with permission), Nature cell biology, Molecular and cell biology (with permission)

Work Area

Here is some bold text

Here is some italic text

First Lab

Journal of cell biology

Lab 2 Questions

1. Which chromosomes contribute to the nucleolus?

Mostly by rRNA genes. The chromosomes 13, 14, 15, 21 and 22, contain nucleolar organiser regions that are located in their short arms.

2. Identify and add a link to your page of a recent cell biology article using confocal microscopy.

An Atlas for Schistosoma mansoni Organs and Life-Cycle Stages Using Cell Type-Specific Markers and Confocal Microscopy.



Lab 3 Questions

1. Find the SDS information for chloroform and identify the hazards of this chemical.

Chloroform SDS

Hazards: This material causes cancer in laboratory animals, and is IARC listed as a probable human carcinogen. Inhalation and ingestion are harmful and may be fatal. May cause reproductive damage. Irritant. Exposure to alcohol may increase toxic effects. Prolonged or repeated skin contact may cause dermatitis. Typical TLV 50 ppm.

2. Upload an image and add it to your page, with the reference and copyright information with the image.

Illustration of a neuromuscular junction

Lab 4 Questions

1. Identify a commercial supplier of an antibody that relates to your group project topic.

Biocompare: Anti-Human Immunoglobulin IgG Monoclonal Antibody, Unconjugated, Clone MH16 / 1 from Cell Sciences IgG (MH16-1) [1]

2. In mitochondria, where is the gene located that encode Cytochrome C and what keeps this protein trapped within the mitochondria?

The gene that encodes Cytochrome C is CYCS gene, it is located on chromosome 7. mitochondria lecture. Cytochrome C is a protein that is located in the inner mitochondrial membrane. It is a water soluble protein and is trapped inside the mitochondria by the outer membrane

Lab 5 Questions

Complete the KO lab written exercise. DONE

Lab 6 Questions

Difference in phenotype groups.jpg

1. What are the changes in phenotypes that you observe between group A and group B in your graph?

Phenotype A: No change in cell numbers between control and TM4 group.

Phenotype B: TM4 group seems to be reduced by 15%, indicating that TM4 may inhibit this cell phenotype.

Phenotype C: There is a small increase in TM4 group about 3% which may not be count in the role that TM4 played in this particular phenotype.

Phenotypes D and E: TM4 groups expressed an increase about 11% and 9% respectively. This may indicate that TM4 is actively involved in the events of neurites growth for these types of phenotypes.

Phenotype F: No cells found for the TM4 group. This may indicate that TM4 entirely inhibits this phenotype.

2. What are the changes in phenotypes that you observe between group A and group B in the pictures?

Genotype A (TM4):

- More branching and processes appear

- This means that there is a considerable interaction between neorites

- The processors seem to be shorter, wider, fluoresce more cheerfully

- Most phenotypes shown were stringed, pronged and stumped

- They emerged in groups (clustered) rarely compared with genotype B

- The nuclei is round, shadowy pink-blue color

- Lamella stained yellow in its edges

- Pink cytoskeleton

Genotype B (control):

- Less branching and processes appear

- Less interaction

- Thinner and often crumbled together

- Most phenotypes shown were broken fan, stumped and stringed

- Very shiny blue nuclei

- The shape of the nuclei appeared in two forms: round and spindle (wide in the middle then tapering at both ends)

- Lamella stained red in its edges

- Red cytoskeleton

Obviously, the morphology of the cell in genotype B unfolded the opposite characteristics compared with the genotype A. This lead to a conclusion that TM4 expressed in genotype A is involved in motility of the cell. These cells showed more branching, processes and hence more interaction.

3. How does TM4 mediate these changes?

Tropomyosin (Tm) is one of the two primary componenets of the actin filaments and the other component is the troponin. Tm contains two polypeptide chains and is a long thin molecule. Tm molecules are bound head to tail, forming a polymer that run over the actin subunits alongside the outer edges of the groove between the twisted actin strands. It binds to actin and acts as a molecular barrier. In relaxed muscle, Tm blocks myosin-binding sites of actin and prevents the crossbridge cycle from occurring. While, the Ca ions released, bind and cause a conformational change to troponin. This shifts tropomyosin’s position on the actin filament and expose the myosin- binding sites.

However, there are several Tm’s isoforms and one of them is Tm4. A striated muscle isoform of a Tropomyosin (TM-4) gene was characterized and found to be necessary for contractile function in embryonic heart. These results demonstrated that a striated muscle isoform of the TM-4 gene was expressed embryonically and was necessary for normal structure and function of the ventricle [2]. Additionally, TM-4 may be involved in the motile events of neurite growth and synaptic plasticity [3]. This is and what we observed for each phenotypes of group A(Tm4) and group B(control). Tm4 group have more processes branching like dendrides tree, which were near each other seeing from cell to cell, allowing more interaction, communications. Other results from new research indicated that Tm-4 plays a role in regulating adhesion structures of osteoclasts, most likely by stabilizing the actin microfilaments present in podosomes and the sealing zone [4].

Lab 9 Questions

1. Identify from one of the cell line repositories: a neural cell line and a muscle cell line.

Neural cell line: CRL-2765 [5] from ATCC [6]

Muscle cell line: CCL-198 [7] from ATCC [8]

2. Identify the species and growth conditions for these cell lines.

Neural cell line: CRL-2765[9]

Species: Rattus norvegicus (rat)

Source: cell type-neuronal Schwann cell; spontaneous immortalization

Morphology: neuronal

Growth Properties: adherent

The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium. To make the complete growth medium, the following components should be added to the base medium: fetal bovine serum to a final concentration of 10%.

Growth condition:

  • Air 95%; carbon dioxide (CO2) 5%
  • 37.0°C (temperature)
  • Remove and discard culture medium.
  • Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  • Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

  • Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
  • Add appropriate aliquots of the cell suspension to new culture vessels.
  • Incubate cultures at 37°C.
  • A subcultivation ratio of 1:6 to 1:10 is recommended
  • Medium renewal, 2-3 times weekly
  • Preservation; freeze medium; complete growth medium supplemented with 5% (v/v) DMSO
  • Storage temperature: liquid nitrogen vapor phase
  • recommended serum:ATCC 30-2020

Muscle cell line: CCL-198 [10] from ATCC [11]

Species: Mus musculus (mouse)

Morphology: fibroblast

Growth Properties: adherent

Source: Tissue- skeletal muscle, Cell type- fibroblast

Growth condition:

  • To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
  • A subcultivation ratio of 1:2 to 1:5 is recommended
  • Medium renewal - twice per week
  • Remove medium, add fresh 0.1% trypsin for about 10 minutes, and centrifuge to remove trypsin. Resuspend cell pellet in fresh medium and dispense into new flasks. Subculture every 7 days.

Peer assessment comments