Biology Department

College of Arts & Sciences Date submitted:

BIOL 117L – Immunology Group No.:
Author: MRB Dacar & NM Odchimar
Members:

EXERCISE 2: FLOW CYTOMETRY AND FLUORESCENCE-ACTIVATED SINGLE

CELL SORTING (FACS)

Flow cytometry is a laboratory method used to detect, identify, and count specific cells (Figure 1). This
method can identify the type of cells in blood or bone marrow sample, and specifically it detects types of
cancer cell based on either the presence or the absence of certain protein markers (antigens) on a cell's
surface. Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It provides a
method for sorting a heterogeneous mixture of cells, one at a time, based upon the specific light scattering
and fluorescent characteristics of each cell.
FACS can detect cell size and complexity, while the fluorescence characteristics allow the researchers to
analyze different biological functions or study specific cell components. It is also based on three main
systems: fluidics, optics, and electronics shown on the schematic flow chart (Figure 2). Their combined
work allows the process of cell sorting. The fluidics system transports particles in a stream to the laser beam
for interrogation. The optics system consists of lasers to illuminate the particles in the sample stream and
optical filters to direct the resulting light signals to the appropriate detectors. The electronics system
converts the detected light signals into electronic signals that can be processed by the computer.

Figure 1. Illustration diagram of Flow Cytometry

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reproduction or any information storage and retrieval system) without written permission from the Biology
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 Figure 2. FACS Schematic Flow Chart.

TASK 1: Watch the provided YouTube links and perform the following tasks.

The Principle of Flow Cytometry and FACS https://www.youtube.com/watch?v=5IdYFgYb9ls&t=403s

The Principle of Flow Cytometry and FACS https://www.youtube.com/watch?v=7bCZx5xPwt0

TASK 2. Perform Flow Cytometry simulation for LEUKEMIA SUBTYPE DIAGNOSIS via
Learn.Genetics

1. Copy and paste this link: https://learn.genetics.utah.edu/content/labs/flowcytometry/ to your own

browser and provide a screenshot (Label as Image 001<FamilyName>).
2. Click LEUKEMIA SUBTYPE DIAGNOSIS and follow the instructions provided.
3. On a separate sheet of paper (for taking notes) write down all the steps in completing this activity.
4. Provide a screenshot image of the flow cytometer result with your corresponding answer (Label
as Image 002<FamilyName>).

This material is owned by the Biology Department – Xavier University and it is for exclusive use. No part of this page
shall be reproduced in any form or by any electronic or mechanical means (including photocopying, recording, CD
reproduction or any information storage and retrieval system) without written permission from the Biology
Department of Xavier University.
TASK 3. Perform Flow Cytometry simulation for MINIMAL RESIDUAL DISEASE via
Learn.Genetics

1. Copy and paste this link: https://learn.genetics.utah.edu/content/labs/flowcytometry/ to your own

browser and provide a screenshot (Label as Image 003<FamilyName>).
2. Click MINIMAL RESIDUAL DISEASE and follow the instructions provided.
3. Provide a screenshot image of the flow cytometer result (Label as Image 004<FamilyName>).

QUESTIONS:
1. What properties of a cell or particle can be measured by a flow cytometer?

2. What are the three main systems in a flow cytometer and its function?

3. What are the applications of Flow Cytometry?

This material is owned by the Biology Department – Xavier University and it is for exclusive use. No part of this page
shall be reproduced in any form or by any electronic or mechanical means (including photocopying, recording, CD
reproduction or any information storage and retrieval system) without written permission from the Biology
Department of Xavier University.
TASK 4. Perform Flow Cytometry with FACS simulation via Labster.
Note: Go through the simulation activity first then proceed in answering the questions below.
1. Copy and paste this link: https://www.biosustain.dtu.dk/Education/Virtual-laboratories/FACS/ to
your own browser.
2. Complete the mission checklist provided for the simulation activity and provide a summary for
each checklist at the end of the activity (use separate sheet).
3. Provide the following screenshots needed (use separate sheet)
(a) Flow Cell Chamber
(b) FACS optics system overview
(c) Negative Control analyses,
(d) Positive Control analyses,
(e) Cell Sorting Preparation: Gating analyses.
(f) Sample and FAC controls overlayed

QUESTIONS:
1. What are the two sections of optic system and its difference.

2. Why GFP is a popular reporter gene?

3. The collection optics detect three different signals, what are they?

4. Why forward scatter (FSC) values can be directly related to cell size?

This material is owned by the Biology Department – Xavier University and it is for exclusive use. No part of this page
shall be reproduced in any form or by any electronic or mechanical means (including photocopying, recording, CD
reproduction or any information storage and retrieval system) without written permission from the Biology
Department of Xavier University.
 5. Why is side scatter (SSC) directly related to cell complexity?

6. Why the mixture needed to be vortexed before adding it to the loading port?

7. What is the purpose of gating?

8. What is the protocol (cleaning order) for shutdown procedure and its purpose?

Reflection/Generalization

END

This material is owned by the Biology Department – Xavier University and it is for exclusive use. No part of this page
shall be reproduced in any form or by any electronic or mechanical means (including photocopying, recording, CD
reproduction or any information storage and retrieval system) without written permission from the Biology
Department of Xavier University.