The Mitochondrial Hub
IMRIC, Faculty of Medicine, Hebrew University of Jerusalem
Hands-On Course: 7-9 January 2014
Measuring Mitochondrial Function and Dysfunction
Organizers: Orian Shirihai & Ann Saada
Faculty: Orian Shirihai, Ann Saada, Ophry Pines, Ajit Divakaruni, David Chess, Maria Torvinen, Ilan Benador,
Gilad Twig, Guy Las, Einav Shnaidman, Avital Swissa
Location: HUJI- School of Medicine, Hadassah Ein Kerem Jerusalem
Botnar lecture facilities, Soren Seminar room
By car; Hadassah Ein Kerem Hospital parking (sorry, no refund)
parking shuttle stop; student cafeteria, descend stairs beside station to Botnar,
Public transport: Tram+Bus 27 from central bus station or bus 19 from downtown, Bus stop: Hadassah Ein Kerem , Medical School
(turn left, cross road to cafeteria, descend stairs to the right to Botnar)
Please print out and bring program to present at security checks
Hands-On Course:
Measuring Mitochondrial Function and Dysfunction
Faculty: Orian Shirihai, Ann Saada, Ophry Pines, Ajit Divakaruni, David Chess, Maria Torvinen, Ilan Benador,
Gilad Twig, Guy Las, Einav Shnaidman, Avital Swissa
Agenda
Tue, January 7, 2013 (Location: Botnar Facility, Soren Seminar room)
Day
Tue
Time
9:00-9:30
Tue
9:30-10:30
Tue
11:00-12:00
Tue
Tue
Tue
Presenting
Orian Shirihai &
Ann Saada
Lecture Title
Describing the workshop, its goals and
introducing, lecturers & trainers
10:30-11:00
~~Break~~
Coffee & Cookies (and carrots)
12:00-13:00
13:00-14:00
Orian Shirihai
~~Lunch~~
A simplified view of the respiratory chain
Provided for workshop attendees
Orian Shirihai
David Chess
Overview: Mitochondrial functions
Overview: Respiratory Chain & the Krebs cycle
Tue
14:00-15:00
Ann Saada
Tue
15:00-16:00
Chess
Tue
16:00-16:30
~~Break~~
Coffee & Cookies
17:30-18:15
Maria
The XF24 and Its Use
Tue
16:30-17:30
Tue
18:15-18:30
Tue
Tue
Tue
18:30-19:15
19:15-20:00
Ajit
~~Break~~
Gilad, Ilan, Ann
Maria
Location
Biochemical measurement of respiratory
chain components
Choosing the right assay for respiratory chain analysis:
Intact cell, isolated mitochondria, perfused cells or
complex activity in cell lysate
Designing Respirometry Experiments for Intact Cells
Coffee & Cookies
Measuring mitochondrial mass: imaging, FACS, protein blots, mtDNA
Principles of Preparing Cells for
Respirometry Experiments
Hands-On Course:
Measuring Mitochondrial Function and Dysfunction
Faculty: Orian Shirihai, Ann Saada, Ophry Pines, Ajit Divakaruni, David Chess, Maria Torvinen, Ilan Benador,
Gilad Twig, Guy Las, Einav Shnaidman, Avital Swissa
Agenda Continued
Wed, January 8, 2013 (Botnar Facility Soren Seminar room)
Day
Wed
Wed
Time
9:00-10:00
10:00-10:45
Presenting
Guy Las
Ann Saada
Topic
Mitochondria, ROS and aging
The principles of Isolating Mitochondria:
methods and quality measures
Wed
10:45-11:00
~~Break~~
Coffee & Cookies (and carrots)
Wed
11:00-12:00
Ajit
Wed
12:00-13:00
Orian Shirihai
Designing experiments for isolated
mitochondria respirometry & permeabilized cells
Analysis of Mitochondrial Membrane Potential
Wed
13:00-14:00
~~Lunch~~
Provided for workshop attendees
Wed
14:00-17:00 Maria, Ajit,
Chess
Location
Wet Lab Work:
The XF software + Running Respirometry Experiments in intact
Adherent Cells in Culture
Location: Med School interdepartmental unit
During the experiment (14:00-15:00) Guy Las will lecture to those who are not participating in the hands on.
Title: Measuring autophagy and autophagic flux
Wed
Wed
17:00-17:45
17:45-18:00
Ilan Benador
~~Break~~
Debugging common artifacts in mitochondrial imaging
Coffee & Cookies
Wed
18:00-19:00
Ajit +Chess
Debugging common artifacts in respirometry
Wed
19:00-20:00 Ajit,
Maria, Chess
Analysis & interpretation of the data from
the cultured intact cells respirometry experiment.
Hands-On Course:
Measuring Mitochondrial Function and Dysfunction
Faculty: Orian Shirihai, Ann Saada, Ophry Pines, Ajit Divakaruni, David Chess, Maria Torvinen, Ilan Benador,
Gilad Twig, Guy Las, Einav Shnaidman, Avital Swisa
Agenda
(Continued)
Thu, February 9, 2013 (Botnar –Facilitiy, 9.00-11.00 Levi Botnar 11.00- 20.00 Soren Seminar room )
Day Time
Thu 9:00-11:00
Presenting
All Faculty
Thu
11:00-11:45
Ajit
Thu
11:45-12:30
Einav Shnaidman
Thu
13:30-15:00
Maria, Ajit, Chess
Wet Lab Work:
Respirometry in PERMEABILIZED cells.
Location: Med School interdepartmental unit
Thu
15:00-16:00
Maria, Chess,
Ajit
Interpretation of permeabilized cells respirometry
Thu
12:30-13:30
~~Lunch~~
Topic
Location
One-on-one session with workshop faculty.
Consulting students about their specific
Mitochondria research needs for their projects
Respirometry with increased physiological relevance:
Hypoxia, 3D models, and substrate oxidation in intact cells
Measuring respiratory function in chunks of tissue:
The fat tissue
Provided for workshop attendees
During the experiment (14:00-14:45) Orian Shirihai will lecture to those who are not participating in the wet lab
experiment. Title: Measuring mitochondrial age and turnover using the MitoTimer. Then, 14:45-15:45 Gilad
Twig, Guy Las and Orian Shirihai will be available to consult on mitochondrial dynamics and autophagic flux
measurements to those who are not participating in the wet lab session.
Thu
16:00-17:00
Gilad Twig
Analysis of mitochondrial
morphology and dynamics
Thu
17:00-17:30
~~Break~~
Coffee & Cookies
Thu
18:30-19:15
Ophry Pines
Is my protein in the mitochondria and how does it get there
Thu
Thu
17:30-18:30
19:15-20:00
David Chess
Orian Shirihai
measuring mitochondrial redox and its interpretation
Integration of mitochondrial membrane
potential and respirometry into the analysis
of a mitochondrial phenotype.