LIFS 5070 (2022)

LIFS 5070 WORKSHOPS IN BIOSCIENCES 2022

Flow Cytometry Section

Flow Cytometry Section:

Faculty-in-Charge: Prof. Joseph T.Y. Wong
Technical officer-in-Charge: Miss Vivian Yu (Rm 6245)

• The aim of this section is to give you some background and hand-on experiences in
flow cytometry, with different staining techniques with different cell types for different
purposes.

• Lecture Session: On 19th Sept (Mon), 12:00-14:00, (Room 5501). This is to introduce
you the principle and application of Flow Cytometry.

•Equipment Introduction Session: Will conduct after the lecture session. In this
session, will briefly introduce students the existing models of flow cytometers we have in
university, the differences among them, the core components of the flow cytometers and
its basic operation.

•Hands-on practice: Conduct on 21st Sept (Wed), 10:00 – 16:00. Students will be
divided to groups of 4 for the practical exercise. Attendance will be taken. Please follow
the protocols attached to prepare samples for all three exercises at Teaching Lab of
Division of Life Science (Room 4160, near Lift 33), and get all samples ready at
indicated time. Please clearly label your ready-to- run samples (particularly with your
group number) and pass to Miss Vivian Yu for flow cytometry analysis.

•Discussion Session: Will be conducted on 27th Sept (Tue),12:00 – 12:30, (Room 4475).
Your flow cytometer analysis results will be distributed in class. In this session, we will
discuss how to interpret the results and what kind of results you supposed to obtain.

Class arrangement summary:

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EXERCISE (1): PHYTOPLANKTON (I) & (II)

Preparing live phytoplankton cells for flow cytometry

Aim. To demonstrate discrimination between phytoplankton species with different sizes

and pigments.
In this case a fuscoxanthin diatom and a peridinin dinoflagellate;
Both have chlorophyll.

N.B. PI is carcinogenic, please wear protective gloves and labcoat at all time!!!

PHYTOPLANKTON (I)
Part I: Phytoplankton cells cover a wide range of size (ranging in size from 0.6 – 60um)
and pigment fluorescence wavelength (usually red and orange). (without staining)
Hope you recall what FSc and SSc represent ??

1. Spin down the different phytoplankton cells (tube 1A to 1E) at 3000rpm for 5 minutes.
2. Discard the supernatant carefully and re-suspend cell pellet in 1 ml of PBS.
3. Transfer samples (1A-D) to 5ml BD Falcon tube provided. Keep on ice bucket. The three
live phytoplankton samples (1A-C) will be characterized based on their sizes, and auto-
fluorescence. Tube 1D is phytoplankton after quenching autofluorescence by solvent
treatment. Tube 1E will be continued to process for part II.
4. pipet small amount (approximately 30ul each sample) of three phytoplankton samples
(sample 1A-1C) onto microscope glass slide and observe their size and morphological
differences under microscope.

PHYTOPLANKTON (II) genome sizes with pigment background

Part II: Propidium iodide (PI) is a commonly used dye for DNA content . One of the
phytoplankton species will be investigated. One of the tubes have not been extracted for
pigments will be provided or done by the technician as negative control.
Aim: the importance of considering the background fluorescence
1. For tube 1E, which is already in 1ml PBS, transfer sample to eppendorf tube; Centrifuge
at 3000 rpm for 5 minutes. Discard the supernatant.
2. Re-suspend cell pellet in 1 ml of 70% Ethanol (fixation step). Incubate on ice for 1 hour.

3. Centrifuge. Discard supernatant. Re-suspend cell pellet with 1ml PBS.

4. Centrifuge again to remove PBS. Re-suspend the cells in 950µl of PBS.

5. Add 50µl of RNaseA (0.5 mg/ml) to the sample;

6. Incubate at room temperature for 30 minutes;

7. To stain DNA, add 25 µl of Propidium Iodide (1 mg/ml) and incubate at room
temperature for 30 minutes, or on ice for longer time. Keep sample in dark.

Transfer samples to 5ml BD Falcon tube for analyze on flow cytometry.

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Reminder: wash all cells with care. One common problem with flow cytometry is the lack of
cells.

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EXERCISE (2): MAMMALIAN CELL (I)

DNA content analysis

In this exercise, the DNA content (mammalian cells) will be investigated by flow cytometry.
The human acute promyelocytic leukemia cell (HL60) was collected in exponentially growth
stage and was fixed in 4% paraformaldehyde. 1x106 cell / ml is provided.

Aim: to demonstrate the simplicity of this most common flow cytometry application
N.B. please wear protective gloves and labcoat at all time!!!

1. Spin down the fixed HL 60 (tube labelled “Exercise 2”) at 2000rpm for 3 minutes.

2. Remove supernatant and re-suspend pellet with 1ml PBS. Transfer sample to Eppendorf
tube.

3. Centrifuge again at 13000 rpm for 15 seconds. Remove supernatant and re-suspend the
cells with 1ml of 0.1% Trion X-100 in PBS. Incubate at room temperature for 3 minutes.

4. Centrifuge again and wash with PBS twice (to remove Triton X-100);

5. Re-suspend cell pellet with 950ul of PBS and 50μl of RNaseA (0.5 mg/ml). Room
temperature incubate for 30 minutes.

6. Add 25 μl of Propidium iodide (1 mg/ml). Incubate at room temperature in dark for 30

minutes, or on ice for longer time.

7. Transfer samples to 5ml BC flow cytometer tube for analysis. Keep the samples at 4C
before analysis.

Reminder: wash all cells with care. One common problem with flow cytometry is the lack of
cells

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EXERCISE (3): MAMMALIAN CELL (II)

Monitoring TPA induced Cell Differentiation

using CD38- a surface antigen marker

Aim: To demonstrate the surface antigen with DNA staining

HL60 is a leukemic progenitor cell line that proliferates and remains in the
promyelocytic stage and fails to differentiate into functional blood cells. Upon addition
of Phorbol 12-myristate 13-acetate (TPA), these cells undergo differentiation along the
granulocytic lineage.

Expression of CD38 antigen is a phenotype that can be induced by TPA. The monoclonal
CD38 antibody provided in this exercise is already fluorochrome (FITC) conjugated.
Only one single antibody labeling step (direct labeling) you can stain your sample.
Unstained control will be provided.

1. Spin down the cells (Tube 3A to 3D) at 1100 rpm for 3 minutes.
Tube (3A): Live HL60 cell, no TPA treatment.
Tube (3B): Live HL60 cell, TPA induced.
Tube (3C): Live HL60 cell, No TPA treatment.
Tube (3D): Live HL60 cell, TPA induced.

2. Resuspend with PBS, the transfer to Eppendorf tubes.

3. Centrifuge at 13000 rpm for 15 seconds. Discard supernatant. Incubate cells in 0.5ml of
0.001% Triton X-100 PBS for 1 minute at room temperature. Be gentle. Not to
unnecessarily damage the cells.

4. Centrifuge again at 13000 rpm for 15 seconds to remove supernatant. Resuspend cell
pellet with 1 ml FACS buffer;

5. Repeat step 4. Discard supernatant.

6. For samples 3A and 3B: Do antibody-staining; Re-susepend cell pellet with 150 μl FACS
buffer. Add 1.5ul of FITC-conjugated anti-CD38 monoclonal antibody to each sample.
Incubate on ice, in dark, for 2 hours.

7. For samples 3C and 3D: As unstained control; Leave in FACS buffer on ice.

7. Spin down the cells of 3A and 3B at 13000 rpm for 15 seconds. Wash cell with 1ml FACS
buffer. Repeat the washing step one more time. Discard the supernatant.

8. Re-suspend cell (3A to 3D) with 0.5ml of FACS buffer, add 0.5ml of 2%
paraformaldehyde; Incubate 30 minutes on ice. Transfer sample to BD flow cytometer tubes.
Keep on ice until analysis.

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