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Deadline:
10th April
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Worth 6 Roberts points

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Programme

mammalian-cell200

Objectives

The main objective of this course is to provide young researchers in biosciences a theoretical and experimental framework enabling them to gain the experience in culturing mammalian cells, inducing or knocking down the expression of cellular proteins, examining subcellular localisation by immunofluorescent microscopy etc.  Although the course will assume basic knowledge of the biomedical sciences, no previous understanding of mammalian cell biology will be required.


All lectures in  meeting room 2, 1st floor Cruciform

All practicals in Cruciform Teaching wet lab G2.01

Tutorial 1 and 2 will take place in Cruciform Dry lab G4.01.  It will also be used as the internet hub for all participants.

Day 1

 
9:00 - 9:30
Registration and coffee, 1st floor meeting room, Cruciform Building UCL
9:30 - 10:00
Welcome address from the organisers
10:00 - 13:00
Practical Session 1:
Part 1:  Culturing and maintenance of mammalian cells (HEK293, U937 and human embryonic stem cells)
During this practical session, the participants will have the opportunity to observe under the light microscope different types of mammalian cells, including HEK293 (human embryonic kidney), U937 (monocytes) and human embryonic stem cells.  The experience will be gained in splitting and counting cells, setting out sterility tests etc.  The techniques of growing cells on the substrate (HEK293) and in suspension (U037) will be taught.
PART 2:  Cryopreservation of mammalian cells
13:00 - 14:30
Lunch  (Cruciform Interactive cafe)
14:30 - 15:30
Lecture 1:  Professor David Saggerson
"Regulation of energy metabolism in mammalian cells"
15:30 - 18:00
Practical Session 2
This session is sponsored and organised by Miltenyi Biotec
PART 1:  Live cell staining of HEK293 cells with plasmid DNA and eGFP mRNA.
Transient transfection of HEK293 cells with plasmid DNA and eGFP mRNA
Transient transfection of HEK293 cells with pEGFP-N1/GFP and pEGFP-N1/GFP-CoA synthase using TurboFect transfection reagent (Fermentas) and eGRP mRNA (Miltenyi Biotec)
 18:30 - 21:00

Reception at UCL

wine and canapes   

Day 2

 
9:00 - 10:00

Lecture 2 Dr L. Ruban

"Human pluripotent stem cell in culture"

 10:00 - 10:30
 Coffee break and discussion
10:30 - 13:00

Practical Session 3
PART 1:  Treatment of cells with Ku-0063794
(mTOR kinase inhibitor, cytostatic), doxorubicin (DNA intercalating drug, cytotoxic), TNFalpha (pro-apoptotic cytokine).  The effect of these drugs/cytokine will be examined in the CellTiter Cell Proliferation Assay.

PART2:  Revival of cryopreserved mammalian cells

13:00 - 14:30
Lunch
14:30 - 15:30

Lecture 3: Prof J. Brockes

"Cell biology underlying vertebrate limb regeneration"

15:30 - 18:00
Tutorial 1: Dr G. Thomas and Prof. I. Gout
The objective of this practical tutorial is to gain an appreciation of the nature of the digital immunofluorescence microscopy image and to experiment with semi-quantification.  It will be carried out in the computer cluster room.  The participants will have the opportunity to gain the experience in using various computer programs in order to compare the intensity of immunofluorescent signals.

Day 3

 
 9:00 - 10:00
Lecture 4:  Dr G. Thomas
"Interactions between cells and their environments: general principles in cell signalling & signal transduction"
10:00 - 10:30
Coffee break and discussion
10:30 - 13:00
Practical Session 4
PART 1:  Testing cell viability and proliferation by the CellTiter Cell Proliferation Assay (initiation of the assay)
PART 2:  Examining the expression of GFP and GFP/CoA synthase fusion proteins in transfected cells by immunofluorescent microscopy.  The participants will compare the transfection efficiency and subcellular localisation of expressed proteins.
13:00 - 14:30
Lunch
14:30 - 15:30
Lecture 5:  Prof I. Gout
"The control of cellular metabolism and growth by signalling pathways"                                            
15:30 - 18:00
Practical Session 5
PART 1:  Completion and analysis of the Cell Proliferation Assay.
The participants will assess the viability and proliferation of cells grown in the presence or absence of Rapamycin (mTOR kinase inhibitor), doxorubicin (cytotoxic, DNA intercalating drug), TNF alpha (tumor necrosis factor-alpha, cytokine) suing the MTT Cell Proliferation Assay.  It is a colormetric assay which offers a quantitative, convenient method for evaluating cellular response to diverse modulators of cell growth and survival.
PART 2:  Summing up tutorial.  The participants will have the opportunity to discuss the application of acquired techniques in experimental set-ups relevant to their research projects.

Coffee in the Historic Housman and presentation of certificates