(Lectures, discussion groups, poster sessions, tutorials & other training activities, practical work if applicable - use page 4a if necessary)

25 January

13.00 – 17.00 Registration, main hall, NaUKMA

17.00 – 17.30 Wellcome address from the organizers

17.30 – 18.30 Keynote lecture: Prof. A. Ridley “The role of small GTPases in mammalian cell biology”

18.30 – 22.00 Dinner and Social (performance by NAUKMA’s traditional folk dancing and music ensembles)

26 January (Monday)

07.30 – 09.00 Breakfast (in the hotel)

09.30 – 10.30 Lecture 1: G. Ritter “Modern approaches for transient and stable expression in mammalian cells”.

10.30 – 11.00 Discussion and coffee break

11.00 – 12.00 Lecture 2. G. Thomas - “Interactions between cells and their environments: general principles in cell signalling & signal transduction”

12.00 – 13.00 Lunch (in Trapezna Academia)

13.00 – 15.15 Culturing and maintenance of mammalian cells. During this practical session, the participants will have the opportunity to prepare media, split and count cells, set up sterility test etc. The technique of growing cells on the substrate (CHO cells, Chinese hamster ovary and RAW264, murine macrophage cell line) and in suspension (hybridoma cells) will be taught. CHO cells will be splitted for transient transfection. In addition, RAW264 cells will be splitted for setting up macrophage differentiation test and hybridoma cells for antibody production.

15.15 – 15.45 Discussion and coffee break

15.45 – 18.00 Practical Session 2: Preparation and analysis of plasmid DNA for transient transfection. Competent cells will be transformed with expression plasmids (pEGFP and pEGFP-FABP4). The transformation efficiency will be analysed next day. To save time, we will inoculate several bacterial colonies into LB broth media from pre-made bacterial cells transformed with pEGFP and pEGFP-FABP4 for overnight culturing.

27 January (Tuesday)

07.30 – 09.00 Breakfast (in the hotel)

09.30 – 10.30 Lecture 3. Prof. S. Sidorenko “Generation of hybrid cells: the application of monoclonal antibodies in research and clinics”.

10.30 – 11.00 Discussion and coffee break

11.00 – 12.00 Lecture 4. Prof. C. Danpure “Whole tissue/cell techniques (immunofluorescence and immunoelectron microscopy, subcellular fractionation, FACS analysis etc)”.

12.00 – 13.00 Lunch

13.00 – 15.15 Practical Session 3: Analysis of pEGFP and pEGFP-FABP4 transformation efficiency. Miniprep purification of plasmid DNA from overnight cultures (mini-prep purification kit from Fermentas will be used during this practical). Spectrophotometric and gel analysis of purified plasmid DNA.

15.15 – 15.45 Discussion and coffee break

15.45 – 18.00 Practical session 4: Induction of macrophage differentiation by mitogenic stimuli will be carried out during this session. The trainees will be taught how to induce differentiate RAW264 cells with insulin and dexamethasone. In addition, transient transfection of mammalian cells (CHO) with purified pEGFP and pEGFP-FABP4 will be carried out. We will employ ExGen500 transfection reagent during this practical.

28 January (Wednesday)

07.30 – 09.00 Breakfast (in the hotel)

09.30 – 10.30 Lecture 5. Prof. Filonenko “Molecular protein techniques (protein isolation & separation, SDS-PAGE, gel chromatography, protein-protein interactions, yeast 2-hybrid screening, immunoprecipitation etc)”.

10.30 – 11.00 Discussion and coffee break

11.00 – 12.00 Lecture 6. Dr. G. Thomas “Studying signal transduction: bridging the gap between molecular structure and cellular function - examples from G-proteins and protein kinases”.

12.00 – 13.00 Lunch

13.00 – 18.00 Tutorial “Computer analysis of immunofluorescence microscopic images”. Prof. C. Danpure

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, which is set up at the new facility at NAUKMA. The participants will have the opportunity to gain the experience in using various computer programs in order to compare the intensity of immunofluorescent signals.

The tutorial session will include:

13.00 – 14.00 Presentation by Prof. C. Danpure on main principles of digital analysis of immunofluorescence images

14.00 – 15.30 Tutorial session 1

15.30 – 16.00 Discussions and coffee break
16.00 – 18.00 Tutorial session 2

Programs requred: Photoshop, Scion, PC Word, Excel

29 January (Thursday)

07.30 – 09.00 Breakfast (in the hotel)

09.30 – 10.30 Lecture 7. Prof. G. Panayotou “ Advanced methodologies in the study of protein interactions”

10.30 – 11.00 Discussion and coffee break

11.00 – 12.00 Lecture 8. Prof. I. Gout “The control of cellular metabolism and growth by signaling pathways”.

12.00 – 13.00 Lunch

13.00 – 15.15 Practical Session 5: The expression of GFP and GFP-FABP4 in transfected CHO cells will be examined by immunofluorescent microscopy. Then participants will lyse transfected (CHO) and differentiated (RAW264) mammalian cells and measure protein concentration in lysates by spectrophotometric analysis. The set up of the immunoprecipitation assay will be taught (using protein A Sepharose and monoclonal antibodies to FABP4)

15.15 – 15.45 Discussion and coffee break

15.45 – 18.00 Practical Session 6: Participants will also prepare the samples of total cell lysate and immune complexes from IP for loading onto pre-cast SDS-PAGE gels. Protein gel electrophoresis will be performed and separated proteins will be transferred from the SDS-PAGE gels onto PVDF membrane (Millipore). After the transfer, the membranes will be blocked with dry milk.

30 January (Friday)

07.30 – 09.00 Breakfast (in the hotel)

09.30 – 10.30 Lecture 9. Prof. P. Coffer “The regulation of proliferation and survival by PI3K-PKB signalling”.

10.30 – 11.00 Discussion and coffee break

11.00 – 12.00 Lecture 10: Prof. I. Vorobjev “Fluorescent microscopy and flow cytometry in cell biology studies”

12.00 – 13.00 Lunch

13.00 – 15.15 Practical Session 7: the membranes will be probed with anti-FABP4 and anti-beta-actin monoclonal antibodies. The immunoreactive signal will be visualized with HRP-conjugated secondary antibody and ECL. The results of the WB analysis will be discussed.

Introduction to hybridoma technology will be pefrormed. As method for positive hybridoma screening ELISA technique will be shown (first step -antigen loading).

15.15 – 15.45 Discussions and coffee break

15.45 – 17.30 Practical Session 8: During this practical the participants will finish the Western-blot technique and analyze the WB image. ELISA will be blocked and primary antibody will be added and left o/n for incubation.

31 January (Saturday)

07.30 – 08.30 Breakfast

09.30 – 11.15 Practical Session 9: ELISA plates will be washed from unbound primary antibody and incubated with HRP-linked antibody. The immune complexes will be stained with ABTS and analyzed by a colorimetric reaction using plate reader scanning.

11.15 – 11.45 Discussions and coffee break

11.45 – 13.00 Summing up tutorial. It would be a good opportunity for the participants to ask questions regarding the techniques learned and to discuss their application in particular research projects.

13.00 – 14.00 Lunch

The lectures of the proposed course will be opened to biology students and research staff of NAUKMA

14.00 Excursion in Kyiv

1 February (Sunday)

Free day and departure