User:Z3288970

From CellBiology

First login post

--z3288970 15:13, 8 March 2012 (EST)

First cell bio lab says hi! --z3288970 15:24, 8 March 2012 (EST)

Group 8 member (with z3254598, z3217686, z3332227)

Lab Attendance

Wk2 --z3288970 15:29, 8 March 2012 (EST)

Wk3 --z3288970 14:11, 15 March 2012 (EST)

Wk4 --z3288970 14:14, 22 March 2012 (EST)

Wk5 ----Z3288970 15:35, 29 March 2012 (EST)

Wk6 -- forgot to sign in, though i was here.

Wk7 ----Z3288970 14:11, 19 April 2012 (EST)

Wk8 --forgot to sign in

Wk9 --Z3288970 14:13, 3 May 2012 (EST)

Wk10 --Z3288970 14:30, 10 May 2012 (EST)

Wk11 --Z3288970 13:54, 17 May 2012 (EST)

Wk12 --Z3288970 14:04, 24 May 2012 (EST)

--Z3288970 14:09, 31 May 2012 (EST)

Lab Lessons

Lab One

(includes quiz)

Lab 1 notes

[Plasma membrane search in PubMed]

[membrane AND mboc4[bookPlasma membrane search in an online textbook]]

Wiki editing help

Lab Two

Lab 2 notes

  • Inverted microscope layout
    • Solves focal length problems, allows special optical techniques
  • Confocal microscopy
    • Laser illumination & Fluorescence emission
    • Pinhole filter in the same focal plane as the sample = “Confocal”
    • 'Scanning' vs 'Spinning Disk' - Methods of scanning of the sample, spinning disk is faster.
    • Multi-illumination - usage of laser illumination allows multiple light sources.
  • Total Internal Reflection Fluorescence microscopy
    • Is very useful to studying the interaction of a cell with a surface
  • Live cell imaging
    • Must keep cell in ‘normal’ conditions
  • Laser Micro-dissection
    • Use a laser and a vacuum to capture/remove cells
  • Super-resolution microscopy
    Superresolution image of the cytoskeleton of a Drosophila epithelial cell.
    • Takes a series of ‘masked images’, slightly shifted to each other, compares them and produces a far higher resolution than the diffraction limit would otherwise allow.

Quiz Two

1. Identify a reference article that uses the "superresolution" microscopy technique.

Superresolution imaging of chemical synapses in the brain.[1]

PMID 21144999

[link to article]

2. What did the paper show that normal microscopy could not show.

Using superresolution microscopy techniques researchers were able to study features down to a nanometer scale, in colour and in 3D. This could not be done with EM techniques nor florescence techniques.

Lab Three

Lab 3 notes


Fixation

  • to reinforce tissue structure
  • to enhance staining
  • to prevent auto-digestion
  • to prevent microbial growth


[database of SDS data]

  • It is important to be well aware of the hazards associated with the chemicals you are working with


General precautions - working with biological samples demands a degree of caution


Methods of Fixation

  1. Fresh frozen
  • Fast, retains protein function and structure & fat
  • Requires specialized equipment, produces thicker sections, tissue may distort while cutting, freezing then thawing degrades tissue.
  • Not preservative
  1. Precipitation
  • using organic solvents
  • fast, preservative, increases cell permeability
  • does not preserve 3d structure, dehydrating
  1. Aldehyde cross-linked
  • Prevents the degradation of protein and nucleic acids - may interfere with protein function and epitopes
  • Paraformaldehyde
    • popular in Immunochemistry
  • Formaldehyde
  • Gluterldehyde
    • Extremely strong fixative

Misc notes

  • Detergents - selectively remove components from the sample
  • Isotonic solutions - ant solution outside this range will introduce artefacts
  • Cell cultures - very easy to fix
  • Paraffin embedding - improved sectioning and preservation
  • Cryoembedding - not suitable for large sections of tissue

Lab Three Quiz

Leukocyte extravasion can be a pathogenic process when signaling goes wrong, such as in cases of multiple sclerosis where there is a recruitment of leukocytes into the CNS where they should not be normally present [2] The presence of excess leukocytes in the CNS blood supply is thought to linked to Disseminated Intravascular Coagulation, or Sepsis[3]

Furthermore, some pathogenic organisms can manipulate or counter the usual signaling processes involved in Leukocyte extravasion an example of which includes Bacilus anthracis more commonly known as Anthrax by which the organism reduces the recruitment of leukocytes to an infection[4]

One of the signalling factors, or chemotactic agents for guiding Leukocyte extravasion is Von Willebrand Factor, released when endothlial cells are injured. [5]

Wk6 lab

Table.PNG

Wk7laba1b1.png

Wk8 lab

Group project: Added a very brief introduction and extended the section on the pathway involved.

Lab 8

[Macrophage; from a tumor induced by Abelson murine leukemia virus ][6]

Musashi-1

"Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. 362 aa" [7] First described in the literature in 1996 [8]


Musashi-1 Rabbit Antibodies


Anti-rabbit IgG with Alexa Fluor® for Immunofluorescence

References

  1. Adish Dani, Bo Huang, Joseph Bergan, Catherine Dulac, Xiaowei Zhuang Superresolution imaging of chemical synapses in the brain. Neuron: 2010, 68(5);843-56 PubMed 21144999
  2. David W Holman, Robyn S Klein, Richard M Ransohoff The blood-brain barrier, chemokines and multiple sclerosis. Biochim. Biophys. Acta: 2011, 1812(2);220-30 PubMed 20692338
  3. J Zhou, M Schmidt, B Johnston, F Wilfart, S Whynot, O Hung, M Murphy, V Cerný, D Pavlovic, C Lehmann Experimental endotoxemia induces leukocyte adherence and plasma extravasation within the rat pial microcirculation. Physiol Res: 2011, 60(6);853-9 PubMed 21995897
  4. Chinh Nguyen, Chiguang Feng, Min Zhan, Alan S Cross, Simeon E Goldblum Bacillus anthracis-derived edema toxin (ET) counter-regulates movement of neutrophils and macromolecules through the endothelial paracellular pathway. BMC Microbiol.: 2012, 12;2 PubMed 22230035
  5. Björn Petri, Andre Broermann, Hang Li, Alexander G Khandoga, Alexander Zarbock, Fritz Krombach, Tobias Goerge, Stefan W Schneider, Claire Jones, Bernhard Nieswandt, Martin K Wild, Dietmar Vestweber von Willebrand factor promotes leukocyte extravasation. Blood: 2010, 116(22);4712-9 PubMed 20716766
  6. W C Raschke, S Baird, P Ralph, I Nakoinz Functional macrophage cell lines transformed by Abelson leukemia virus. Cell: 1978, 15(1);261-7 PubMed 212198
  7. T Nagata, R Kanno, Y Kurihara, S Uesugi, T Imai, S Sakakibara, H Okano, M Katahira Structure, backbone dynamics and interactions with RNA of the C-terminal RNA-binding domain of a mouse neural RNA-binding protein, Musashi1. J. Mol. Biol.: 1999, 287(2);315-30 PubMed 10080895
  8. S Sakakibara, T Imai, K Hamaguchi, M Okabe, J Aruga, K Nakajima, D Yasutomi, T Nagata, Y Kurihara, S Uesugi, T Miyata, M Ogawa, K Mikoshiba, H Okano Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell. Dev. Biol.: 1996, 176(2);230-42 PubMed 8660864
  9. Hideyuki Okano, Hironori Kawahara, Masako Toriya, Keio Nakao, Shinsuke Shibata, Takao Imai Function of RNA-binding protein Musashi-1 in stem cells. Exp. Cell Res.: 2005, 306(2);349-56 PubMed 15925591
  10. S Sakakibara, T Imai, K Hamaguchi, M Okabe, J Aruga, K Nakajima, D Yasutomi, T Nagata, Y Kurihara, S Uesugi, T Miyata, M Ogawa, K Mikoshiba, H Okano Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell. Dev. Biol.: 1996, 176(2);230-42 PubMed 8660864
  11. Christopher S Potten, Catherine Booth, Gregory L Tudor, Dawn Booth, Gerard Brady, Patricia Hurley, Gary Ashton, Robert Clarke, Shin-ichi Sakakibara, Hideyuki Okano Identification of a putative intestinal stem cell and early lineage marker; musashi-1. Differentiation: 2003, 71(1);28-41 PubMed 12558601
  12. Robert B Clarke, Katherine Spence, Elizabeth Anderson, Anthony Howell, Hideyuki Okano, Christopher S Potten A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev. Biol.: 2005, 277(2);443-56 PubMed 15617686


Lab 9

--Mark Hill 13:31, 17 May 2012 (EST) You have not completed the peer assessment process yet. If you have made comments on each project page they need also to be pasted here today for me to include in your individual assessment.

Lab 12 - Microarray

   Identify a current technique used in gene sequencing.
   Identify a recent cell biology research paper that has used microarray technology.
   What aspect of the research findings were contributed by the microarray technique.
  1. High-thoroughput DNA sequencing: By parallelising the DNA squencing process, new generation machines are able to vastly increase their thoroughput.
  1. Identification of MicroRNAs Inhibiting TGF-β-Induced IL-11 Production in Bone Metastatic Breast Cancer Cells[1]
  1. Microarrays were used in the aforementioned study to assess the type and levels of miRNA present in the samples produced in the study.
    1. Sirkku Pollari, Suvi-Katri Leivonen, Merja Perälä, Vidal Fey, Sanna-Maria Käkönen, Olli Kallioniemi Identification of microRNAs inhibiting TGF-β-induced IL-11 production in bone metastatic breast cancer cells. PLoS ONE: 2012, 7(5);e37361 PubMed 22629385