Methodologies used to analyse cell function.ppt

2nd lecture of cell culture, ELISA and FACS.

1. Cell Structure &
Function

2. Week 11 learning objectives
•Be able to provide a high-level overview of analytical
techniques and methods that scientists use to study
cellular function
•Understand the principles behind ELISA, FACS and
Mammalian culture

3. Lecture 13 learning objectives
•Be able to describe mammalian cell culture
principles and equipment required.
•Briefly describe the history and importance of
mammalian cell culture

4. Basic Introduction to Cell Culture
https://youtu.be/RpDke-Sadzo?si=g73jDwa3LjzvMOAD

5. Why is Cell Culture important?
•Mammalian cell culture is an
important tool for research,
clinical, and pharmaceutical
applications. Cells isolated
from animal tissues can be
expanded in culture to study
cell biology and disease or
used for the production of
antibodies, proteins, and
vaccines

6. Next Year INTRA…after Uni?

7. Next Year INTRA…after Uni?
Search…Nov 2023

9. •From the ancient Romans, through the Middle Ages, to the late of
the nineteenth century, the Aristotelian doctrine of spontaneous
generation was one of the most basic laws in biological sciences.
This idea was presented for the first time by Aristotle in his History
of Animals, where he described the generation of insects from
animal flesh, mud, and other organic and inorganic matter [27].
According to this thesis, non‐living matter (water, land or hay) bears
the potential to generate spontaneously different and complex
organisms. For example, in the seventeenth century literature,
recipes for mice were known—the mixture of old shirts and wheat
placed in a jar for 21 days produced mice. Even the invention of the
microscope and investigations of Leeuwenhoek and Hook did not
refute the Aritostelian doctrine.
The end of spontaneous
generation
https://www.intechopen.com/chapters/
53566

10. •Technical improvements in microscope constructions helped in 1838
the botanist Matthias Schleiden (1804–1881) and in 1839 the
zoologist Theodor Schwann (1810–1882) to formulate the “cell
theory”. They suggested that every organism and every structural
element of plant and animal tissues are formed of cells.
The cell theory
https://www.intechopen.com/chapters/
53566

11. •The American embryologist Ross Granville Harrison (1870–1959)
developed the first techniques of cell culture in vitro in the first
decade of the twentieth century. In Harrison's experiments (1907–
1910, at the Yale University), small pieces of living frog embryonic
tissue were isolated and grew outside the body. He placed the tissue
in a solution of lymph on a coverslip, inverted the material on a
glass slide with a depression in it and maintained the explanted
tissue in a hanging drop.
Harrison’s hanging drop technique and Dr. Carell’s
immortal cells
https://www.intechopen.com/chapters/
53566

12. •In January, 1912, Alexis Carrel (1873–1944) and his coworkers
developed the first “cell line” derived from the fragments of
explanted chicken embryo heart. This cell line was subcultured
hundreds of times, and after the initial contamination outbreak, it
was continued by Arthur Ebeling in Carrel's laboratory. This cell line
was maintained by washing with Ringer's solution and medium
changes. Due to the rigorous aseptic techniques, this is one of the
most famous cell lines (described in many articles, e.g., cell line
birthday was celebrated annually in the New York World Telegram);
it was maintained until 1946 when the cell line culture was finally
terminated, 2 years after Carrel's death.
Harrison’s hanging drop technique and Dr. Carell’s
immortal cells
https://www.intechopen.com/chapters/
53566

13. In the first decade of the twentieth century, Ross Harrison developed
the first techniques of cell culture in vitro, and Burrows and Carrel
improved Harrison's cell cultures. In mid‐twentieth century, the basic
principles for plant and animal cell cultures in vitro were developed,
and human diploid cell lines were established. On the basis of
knowledge about the cell cycle and gene expression regulation, the
first therapeutic proteins were produced using mammalian cell
cultures. The end of twentieth century and early twenty‐first century
brought the progress in 3‐D cell culture technology and created the
possibility of the tissue engineering and the regenerative medicine
development.

15. Senescence: loss of a cells ability to divide and grow, a process of
deterioration with age

17. ? ? ?

20. Why not just do cell culture on the
bench?

21. What not just do cell culture on the
bench?

22. Cell Culture Equipment
BE CAREFUL
Laminar flow hood v
fume hood?
Fume hoods prevent
hazardous
substances from
exiting the hood
workspace, whereas
laminar flow cabinets
prevent contaminants
from entering the
cabinet workspace.

23. Cell Culture Equipment

24. Good Cell Culture practice

25. Lecture 13 learning outcomes
•History and importance of cell culture
•Principles of cell culture including cell types
and growth requirements
•Equipment and procedures to ensure sterile
cell culture

26. Cell Structure &
Function

27. Lecture 14 learning objectives
•Be able to describe the main components of an
enzyme-linked immunosorbent assay and the
basic principles of how it works.
•Be able describe how ELISA can be used to
measure cell function and communication

28. Cell Communication (from cultured
cells)
How can scientists study cell
communication of cultured
cells?

29. Cell Communication (from cultured
cells)How can scientists study cell communication of
cultured cells?
Answer Method 1: ELISA
Scientist can measure the cell signaling molecules
secreted by cells using an Enzyme Linked
ImmunoSorbent Assay (ELISA)

30. Introduction to ELISA (basic level)
There are many different types of ELISA, but for this BE230
course we are going to just focus on one type, the Sandwich
ELISA and it use in cell communication studies.

31. Antibodies are the key element of
ELISA

32. Antibodies are the key element of
ELISA
Antibodies have the ability to bind (recognise) a wide range of molecules
(antigens). One antibody will bind one type of antigen (specificity) and can bind
at high affinity (sensitivity). Therefore, if a good high affinity antibody specific for
a cell signaling cytokine is obtained, then it can be used in an assay to identify
low amounts of that particular cell signaling cytokine. The assay will be specific
for that cytokine e.g. TNF alpha and the ELISA will be called an TNF alpha
detection ELISA.

33. Introduction
•Enzyme immunoassays are techniques developed to detect and
quantify antigens or antibodies in a sample
•Both involve labelling (usually) the antibody with an enzyme which
can be used to convert a substrate to an easily detectable product
which allows for determination of the amount of antibody/antigen
present
•There are two classes of enzyme immunoassays
•Heterogeneous assays involve the immobilisation of one of the components
of the system and separation of free and bound antibody/antigen
•Homogeneous assays do not require separation of free and bound
components

34. Enzyme-Linked ImmunoSorbent
Assay (ELISA)
•ELISA is an example of a heterogeneous assay, in which the
antibody is labelled with an enzyme (such as Horseradish
Peroxidase) which converts a colourless substrate to a
coloured product to allow for detection
•There are several different formats of ELISA, of varying
degrees of complexity, each of which is suitable for a different
type of sample or assay
•The surface used for immobilisation is normally a plastic plate
such as Nunc™ Microtitre plates, which contain 96 individual
assay wells

36. Sandwich ELISA
•Two antibodies to two different epitopes

37. Sandwich ELISA

38. Sandwich ELISA

39. Sandwich ELISA types

40. •What shape is a sandwich ELISA curve and why?
W
O
R
K
T
O
G
E
T
H
E
R

41. Sandwich ELISA

42. Sandwich ELISA E.g. Kit

43. Cell Communication (from cultured
cells)How can scientists study cell communication of
cultured cells?
Answer Method 1: ELISA (Summary)
Testing of the cell culture
media/supernatant from cells
exposed to different study
conditions and to different time
points

44. Lecture 14 learning outcomes
•Explain how antibodies are critical for ELISA
•Principles of Sandwich ELISA and steps
involved
•How ELISA can be used to study cell
communication and function

45. Cell Structure &
Function

46. Lecture 15 learning objectives
•Be able to describe the principles of FACS.
•Be able to describe how FACS is used for
studying cellular function.

47. Cell Communication (from cultured
cells)How can scientists study cell communication of
cultured cells?
Answer Method 2: FACS
Fluorescence-activated cell sorting (FACS),
sometimes called fluorescence-assisted cell
sorting, is a specialized type of flow
cytometry that uses fluorescent markers to
target and isolate cell groups. This cell
sorting technique is commonly used in blood
cell analysis, oncology, and stem cell biology
research.

48. Cell Communication (from cultured
cells)How can scientists study cell communication of
cultured cells?
Answer Method 2: FACS
Flow cytometry and cell sorting by FACS. In
the flow cell (1), the single file of cells passes a
laser and an array of detectors (2), that
measure cell size (with forward scatter, FSC),
cell granularity/aggregation (with side
scatter,SSC) and fluorescence. When the cells
exit the flow cell though the nozzle in
individual droplets (3), they are either or not
given an electrical charge. Droplets with
desired cells that possess a charge will be
bend out of the downward going stream by
electromagnets (4) into collection tubes on
the side (5). The uncharged cells simply drop
straight down into a waste collection tank.

49. Cell Communication (from cultured
cells)How can scientists study cell communication of
cultured cells?
Answer Method 2: FACS
Fluorescence-activated cell sorting
(FACS), sometimes called fluorescence-
assisted cell sorting, is a specialized type
of flow cytometry that uses fluorescent
markers to target and isolate cell
groups. This cell sorting technique is
commonly used in hematopoiesis,
oncology, and stem cell biology
research.

50. FACS introduction (basic)
Which method should I use, FACS or ELISA?
Answer: ELISA is great for measuring signals and molecules
secreted by the cell (the secretome) in the culture media.
FACS is used to measure the cells themselves and can
measure changes to receptors and other molecules on the
surface of the cell.
Answer: They both use antibodies specific for the molecule
of interest as the biorecognition element in the assay i.e.
FACS and ELISA can use a range of antibodies specific for
different markers to study multiple cell communication and
function attributes.
What do FACS and ELISA have in common?

51. FACS introduction (basic)
https://youtu.be/Ut9f_d19wPc?
si=lmirvcMf15jaCnEW

52. FACS introduction (basic)
Cell preparation – what do you want to measure
etc

53. FACS introduction (basic)
Cell preparation – what do you want to measure etc
Depending on the protocol used, FACS can be used to measure both
extracellular and intracellular targets
Fixation with chemicals such as paraformaldehyde allows for cell samples to be stable for longer
periods of time, which provides users with greater control and flexibility in planning their flow
cytometry assays.

54. FACS introduction (basic)
Cell preparation – what do you want to measure etc
https://www.ptglab.com/support/flow-cytometry-protocol/flow-cytometry-sample-
preparation/

55. FACS introduction (basic)
How does flow cytometry work?
https://www.ptglab.com/support/flow-cytometry-protocol/flow-cytometry-sample-
preparation/
1.   First, a cell suspension solution flows
through the chamber where it is then
separated into a stream of single cells by
the fluidics system.
2. The solution then passes through a laser
beam or a set of lasers (Figure 1 – flow
cytometry overview).
3.   A set of lenses, filters, and dichroic
mirrors are used to collect and separate the
light.
4. The light is collected at a 90-degree angle
(side scatter – SSC) and directly opposite the
cell flow (forward scatter – FSC).
5. The light is detected using photodiodes or
photomultiplier tubes (PMTs).
Besides these inherent physical properties,
fluorescence from markers can be assayed in
a similar manner. Some flow cytometers can
also sort cells based on the user gating
conditions of these properties. This type of
flow cytometry is often referred to as
fluorescence-activated cell sorting (FACS).

56. FACS introduction (basic)
How does flow cytometry work?
https://www.ptglab.com/support/flow-cytometry-protocol/flow-cytometry-sample-
preparation/
1.   First, a cell suspension solution flows
through the chamber where it is then
separated into a stream of single cells by the
fluidics system.
2. The solution then passes through a
laser beam or a set of lasers (Figure 1 –
flow cytometry overview).
3.   A set of lenses, filters, and dichroic
mirrors are used to collect and separate the
light.
4. The light is collected at a 90-degree angle
(side scatter – SSC) and directly opposite the
cell flow (forward scatter – FSC).
5. The light is detected using photodiodes or
photomultiplier tubes (PMTs).
Besides these inherent physical properties,
fluorescence from markers can be assayed in
a similar manner. Some flow cytometers can
also sort cells based on the user gating
conditions of these properties. This type of
flow cytometry is often referred to as
fluorescence-activated cell sorting (FACS).
Laser can excite
fluorescent probes on
cells

57. FACS introduction (basic)
How does flow cytometry work?
https://www.ptglab.com/support/flow-cytometry-protocol/flow-cytometry-sample-
preparation/
1.   First, a cell suspension solution flows
through the chamber where it is then
separated into a stream of single cells by the
fluidics system.
2. The solution then passes through a laser
beam or a set of lasers (Figure 1 – flow
cytometry overview).
3.   A set of lenses, filters, and dichroic
mirrors are used to collect and separate
the light.
4. The light is collected at a 90-degree angle
(side scatter – SSC) and directly opposite the
cell flow (forward scatter – FSC).
5. The light is detected using photodiodes or
photomultiplier tubes (PMTs).
Besides these inherent physical properties,
fluorescence from markers can be assayed in
a similar manner. Some flow cytometers can
also sort cells based on the user gating
conditions of these properties. This type of
flow cytometry is often referred to as
fluorescence-activated cell sorting (FACS).

58. FACS introduction (basic)
How does flow cytometry work?
https://www.ptglab.com/support/flow-cytometry-protocol/flow-cytometry-sample-
preparation/
1.   First, a cell suspension solution flows
through the chamber where it is then
separated into a stream of single cells by the
fluidics system.
2. The solution then passes through a laser
beam or a set of lasers (Figure 1 – flow
cytometry overview).
3.   A set of lenses, filters, and dichroic
mirrors are used to collect and separate the
light.
4. The light is collected at a 90-degree
angle (side scatter – SSC) and directly
opposite the cell flow (forward scatter –
FSC).
5. The light is detected using photodiodes or
photomultiplier tubes (PMTs).
Besides these inherent physical properties,
fluorescence from markers can be assayed in
a similar manner. Some flow cytometers can
also sort cells based on the user gating
conditions of these properties. This type of
flow cytometry is often referred to as
fluorescence-activated cell sorting (FACS).

59. FACS introduction (basic)
How does flow cytometry work?
https://www.ptglab.com/support/flow-cytometry-protocol/flow-cytometry-sample-
preparation/
1.   First, a cell suspension solution flows
through the chamber where it is then
separated into a stream of single cells by the
fluidics system.
2. The solution then passes through a laser
beam or a set of lasers (Figure 1 – flow
cytometry overview).
3.   A set of lenses, filters, and dichroic
mirrors are used to collect and separate the
light.
4. The light is collected at a 90-degree angle
(side scatter – SSC) and directly opposite the
cell flow (forward scatter – FSC).
5. The light is detected using photodiodes
or photomultiplier tubes (PMTs).
Besides these inherent physical properties,
fluorescence from markers can be assayed in
a similar manner. Some flow cytometers can
also sort cells based on the user gating
conditions of these properties. This type of
flow cytometry is often referred to as
fluorescence-activated cell sorting (FACS).
Typical flow cytometer instruments use
photomultiplier tubes (PMTs) to detect
the fluorescence in the samples. The
initial signal (light hitting the detector) is
amplified within the PMT to improve the
sensitivity and resolution of the
measurements.

60. FACS introduction (basic)
How does flow cytometry work?
https://www.ptglab.com/support/flow-cytometry-protocol/flow-cytometry-sample-
preparation/
1.   First, a cell suspension solution flows
through the chamber where it is then
separated into a stream of single cells by the
fluidics system.
2. The solution then passes through a laser
beam or a set of lasers (Figure 1 – flow
cytometry overview).
3.   A set of lenses, filters, and dichroic
mirrors are used to collect and separate the
light.
4. The light is collected at a 90-degree angle
(side scatter – SSC) and directly opposite the
cell flow (forward scatter – FSC).
5. The light is detected using photodiodes or
photomultiplier tubes (PMTs).
Besides these inherent physical properties,
fluorescence from markers can be assayed in
a similar manner.
6. Some flow cytometers can also sort
cells based on the user gating conditions
of these properties. This type of flow
cytometry is often referred to as
fluorescence-activated cell sorting (FACS).

61. FACS introduction (basic)
What does the output data look like?
https://www.abcam.com/content/data-analysis-in-flow-cytometry
Flow cytometry data is typically
represented in one of two ways:
histograms, which measure or compare
only a single parameter, and dot-
plots which compare 2 or 3 parameters
simultaneously on a two- or three-
dimensional scatter-plot.

62. FACS introduction (basic)
What does the output data look like?
https://www.abcam.com/content/data-analysis-in-flow-cytometry

63. FACS introduction (basic)
What does the output data look like?
https://www.abcam.com/content/data-analysis-in-flow-cytometry

64. FACS introduction (basic)
(Recap) What applications can it be used for?

65. Lecture 15 learning outcomes
•Explain how antibodies are critical for FACS
•Principles of FACS and steps involved
•How FACS can be used to study cell
communication and function