User:Z3375627

From CellBiology

Z3375627

Lab Attendance

Individual Assessment

Lab 1

Pubmed


Cells Eukaryotes and Prokaryotes

PMID 25513760

<pubmed>25513760</pubmed>


Localization of prokaryotic protein expression.jpg

Localization of prokaryotic protein expression [1]


Lab 2

In 2014, Kevin Takasaki and Bernardo L. Sabatini, conducted a study on whether the time-course of a passive diffusional equilibrium across the spine neck of a dendritic spine neck was determined by its structure. Previous optical technique, 2-photon laser scanning microscopy (400nm), was limiting image resolution of dendrite spine necks, having a a diameter of around 100nm. using stimulated emission depletion (STED), fluorescence imaging of neurons was allwed to a resolution of around 50nm within brain tissue, making it viable to distinguish the fine morphology that could predict the synaptic signalling of the spine

Brain tissue sliced from the hippocampus of mice was prepared and dye filled and then photobleached. Using this combination of 2PLSM and STED, the STED-2P microscope was able to make a morphological analysis and observe diffusional transfer across the spine neck. Although the study demonstrated that more complex geometric models may be required in the instance of those particular dendrites, the use of STED-2P in nanoscale visualisation of other biological systems could be of huge potential [2]

<pubmed>24847215</pubmed>

Lab 3

Paraformaldehyde msds

http://sydney.edu.au/medicine/medsci/whs/msds/p/paraformaldehyde__Sigma__20130416.pdf


Fibronectin wound healing.jpg

Fibronectin Wound healing [3]

Reference Searching


<pubmed>20501851</pubmed>

Plasma Fibronectin (pFN) can be found to be upregulated in tumour cells, supporting tumor retention. Tumor cell adhesion to and invasion in fibrin when paired with fibronectin (fibFN) is mediated mainly by integrin avB3 and activated by the former. fibFN however had no effect on the cell tumor growth. pFN in accociation with fibrin is shown to assist in the cell adhesion of clotted plasma. pFN does not assist in the initial tumor cell arrest.[4]

<pubmed>23356939</pubmed> Sheep Carotid ateries were used to test the coating of proteins on Extracorporeal membrane oxygentators to overcome it's limitations with hemocompatibility, the activation of the coagulation and complement system as well as plasma leakage and protein deposition. Fibronectin increased cell attachment compared to other methods (including uncoated) on ECMO oxygenator membrane. 93% FN cells seeded adhered on treated surfaces after 24hrs compared to 73% on control. adherance occured for 4 days which leads to the discussion of endothelialisation of ECMO membranes to make them more suitable for long term use. [5]



<pubmed>25180602</pubmed>

Plasma fibronectin is found to deposit in vessal injury sites (independant of fibrinogen, von Willebrand factor, β3 integrin, and platelets) before before acummulation of platelets which was previously understood to be the first wave of hemostasis. Promotes platetlet aggregation when in conjunction of fibrin, but acts conversely when fibrin is absent. Working off the fibrin gradient, fibronectin is a regulator of thrombosis[6]


<pubmed>22514136</pubmed> A high fat diet was experiment in mice was conducted in order to cultivate atherosclerotic lesion and the effect the lack of plasma fibronectin would have on the formation of these lesions. Mx-Cre-mediated deletion of the the FN gene was found to reduce atherosclerosis formation.

Plasma Fibronectin is found to help and hinder atherosclerosis. It increases the number and size of Atherosclerosis plaques that form with it's deposition within lesions, but also helps in the formation of a protective fibrous cap, helping prevent rupture of said athersclerotic plaque. Also indicated that plasma fibronectin, rather than the hemopoietic cell-driven fibronectin, is deposited at atherosclerosis-prone sites prior to the development of atherosclerotic lesions.[7]

Lab 4

Knockout mice lab worksheet

Lab 5

Tm4 against control.PNG

Lab 6

Identify an antibody that can been used in your group's extracellular matrix project. Anti-Fibronectin antibody [A17] (ab26245) - This antibody inhibits integrin-mediated cell adhesion to the cell binding domain of fibronectin. It can be used to probe fibronectin conformation. Strong reaction is seen in ELISA with thrombospondin directly coated onto the microtiter well.

Identify the species deriving the antibody. Mouse monoclonal [A17] to Fibronectin

Identify the working concentration for the antibody. stored at 100 µg at 1 mg/ml

1/100 for Western Blotting

1/30000 for enzyme-linked immunosorbent assay

IP, IHC-P, IGC-Fr, ICC/IF: Use at an assay dependent concentration.

Identify a secondary antibody that could be used with this antibody. Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)

Identify a paper that has used this antibody. <pubmed>1385458</pubmed>[8]


Lab 7

no individual assessment

Lab 8

Lab 9

Cell line databases

  • ECACC


human: Hybridoma Collection: W6/1

https://www.phe-culturecollections.org.uk/products/celllines/hybridoma/detail.jsp?refId=13082201&collection=ecacc_hc

Mouse: Hybridoma Collection: OX-80

https://www.phe-culturecollections.org.uk/products/celllines/hybridoma/detail.jsp?refId=13082211&collection=ecacc_hc


  • ATCC


Human: 293T/17 [HEK 293T/17] (ATCC® CRL-11268™)

http://www.atcc.org/Products/All/CRL-11268.aspx

human cell line culture medium: Dulbecco's Modified Eagle's Medium (DMEM) (ATCC® 30-2002™). To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.

 http://www.atcc.org/products/all/30-2002.aspx

medium formulation: http://www.atcc.org/~/media/D7399222FD8B4EF68FD56DD8058804BA.ashx

Mouse: MPRO Cell Line, Clone 2.1 (ATCC® CRL-11422™)

http://www.atcc.org/Products/All/CRL-11422.aspx


penicillin streptomycin This antibiotic mixture contains penicillin and streptomycin. Penicillin is an anti-bacterial agent produced by Penicillium. It interferes with the final stage of bacterial cell wall synthesis, the cross-linking of different peptidoglycan strands. Streptomycin is an anti-bacterial agent produced by Streptomyces. It binds to the 30S subunit of the bacterial 70S ribosome and blocks the initiation complex of protein synthesis. The antimicrobial spectrum for Penicillin- Streptomycin Solutions includes gram-positive and gram-negative bacteria.

Lab 10

Lab 11

Lab 12

References

  1. <pubmed>23951183</pubmed>
  2. <pubmed>24847215</pubmed>
  3. <pubmed>21923916</pubmed>
  4. <pubmed>20501851</pubmed>
  5. <pubmed>23356939</pubmed>
  6. <pubmed>25180602</pubmed>
  7. <pubmed>22514136</pubmed>
  8. <pubmed>1385458</pubmed>

Group Assessment