fluorescence 3

Question 1

What does flow cytometry allow us to do?

Answer 1

1.the physical characteristics (e.g., cell size, granularity)
2. multiple fluorescent parameters of cells

at single cell level

Question 2

FC is capable of high-speed detection, quantitation & multi-parameter phenotyping of individual cells.

true or false

Answer 2

True

Question 3

What are the three main constituents of flow cytometer setup?

Answer 3

1. fluidic system
2. detection/optics system
3. sorting system

Question 4

In FC, the sorting system consists of ___________________.

Answer 4

deflection pates and collection tubes

Question 5

What is the purpose of flow cell in FC?

Answer 5

deliver cells to center of sheath stream

Question 6

What is ‘sheath fluid’ in FC?

Answer 6

fluid within which cells are running in co-axial flow file

Question 7

What is the purpose of deflection plates in FC?

Answer 7

metal plates that carry a high voltage used to deflect drops for sorting

Question 8

Light scattered in what angle is usally measured as SSC in FC?

Answer 8

90 degrees

Question 9

Why does the pressure of cell sample be higher than pressure of sheath fluide during injection from flow-cell into sheath fluid?

Answer 9

1. Ensures that the sample is injected into the sheath
   fluid.
2. Maintains laminar flow and prevents turbulence

Question 10

What is the sheath fluid?

Answer 10

Isotonic buffered solution

Question 11

What is the purpose of sheath fluid in FC?

Answer 11

to achieve Hydrodynamic Focusing in flow cytometer

Question 12

Why is it important to acheive hydrodynamic focusing in FC?

Answer 12

• To generate a laminar flow without turbulence.
• To thin out cell suspension.
• To guide single cells in a single line to a focal point.
• Cells passing thro’ the laser beam one at a time.
• Allows the cells to be measured discretely

Question 13

During the analysis of WBC in blood, hhow are red blood cells lysed?

Answer 13

RBC Lysing Buffer

–> NH4Cl (8.02 g), KHCO3 (1 g) in 800 mL H2O for 30 min

Question 14

How does the RBC lysing buffer lyse RBCs?

Answer 14

NH4Cl acccumulation –> increase in osmotic pressure and pH –> lysis

RBC more susceptible than other cells to changes in osmotic pressure and pH

Question 15

Some some WBCs

Answer 15

Basophil, Eosinophil, Neutrophil, Monocyte, Lymphocytes

Question 16

What kind of information does SSC provide?

Answer 16

info abt internal complexity (granularity) of cell

cellular components that increase SSC –> granules and nucleus

Question 17

What kind of information does FSC provide?

Answer 17

discrimination of cells by size
–> can help distinguish between cells of immune system

Question 18

The intensity of FSC is proportional to the diameter of the cell.

True or False

Answer 18

True

Question 19

State what happens in blood count of lymphocyts, monocytes and granulocytes during normal state, viral infection and bacterial infection

Answer 19

normal sample: Granu > lymp&raquo_space; mono

Viral infection: surge in lymp&raquo_space;> granu&raquo_space;> mono

bacterial infection: surge in Granu&raquo_space;> lymp&raquo_space;> mono

Question 20

What angle Forward Light Scatter (FSC) is detected in the forward direction?

Answer 20

160-200

Question 21

Rank the WBCs in terms of increasing cellular complexity and thus increasing SSC: lymphocytes, monocytes and granulocytes (neutraphils)

Answer 21

lymphocytes &laquo_space;monocytes &laquo_space;granulocytes(neutraphils)

Question 22

In FC, what may the curve shape be affected by?

Answer 22

Flow Rate, Sample Pressure etc

Question 23

What are the 2 major to-dos during data analysis of FC?

Answer 23

1. threshold
2. gating

Question 24

What is meant by threshold?

Answer 24

To discard useless information from cell fragments

Question 25

What is meant by gating?

Answer 25

A selected region for the flow data to be included in subsequent analysis or sorting --> target densest area of graph --< enhanced accuracy and reliability

Question 26

What are the 2 types of T cells that could be studies thru phenotypic marker study in FC?

Answer 26

CD4 + Helper T Cells and CD8 + Cytotoxic T Cells Double Negative Thymocytes differentiate into Double Positive Thymocytes (CD4 + & CD8 +) differentiate into T cells double neg --> double pos --> CD4 or CD8

Question 27

What is meant by CD in say 'CD4 helper T cells'?

Answer 27

Cluster of Differentiation --> cell surface marker on leukocytes --> for the identification & characterization of different subpopulations of leukocytes

Question 28

What kind of cells always increase Non-specific Bindings & Autofluorescence? How to overcome this problem?

Answer 28

Dead cells --> include viability control

Question 29

Change in CD4 & CD8 T-cells in a Patient with AIDS ?

Answer 29

HIV specifically infects and destroys CD4+ T-cells --> decline in CD4 T cells CD8+ T-cells (or cytotoxic T-cells) are responsible for killing virus-infected cells --> initial increase and then functional exhaustion

Question 30

What are the phases in cell division?

Answer 30

G1 phase S phase G2 and M phase

Question 31

In FC, what is used to stain DNA?

Answer 31

Propidium Iodide

Question 32

Describe the process of staining DNA in FC

Answer 32

* PI cannot pass through intact cell membranes in viable cells * Fixed the cells & permeabilized the cell membrane with alcohol * Use RNase to lyse RNA * Add PI to label DNA

Question 33

At what phase (of cell division) are most cells found in?

Answer 33

G1 phase >> G2 and M phase>> S phase

Question 34

In FC, can we measure Ca2+ changes? how?

Answer 34

Yes by using Fluo-4

Question 35

Name some purposes of doing analysis using FC

Answer 35

1. phenotypic marker study (eg lymphocytes) 2. cell cycle analysis 3. cell sorting (to collect useful cells) 4. studying size/complexity of cells

Question 36

Describe the process of cell sorting using FC (FACS)

Answer 36

1. droplet charged after measurement of fluorescence 2. deflected by deflection plate into collection tube

Question 37

What is an important parameter that has to be considered in FASC?

Answer 37

feedback loop to determine whether cell meet sorting criteria then known delay time: delay between fluorescence detection (at the laser interrogation point) and the droplet reaching the sorting location

Question 38

What are some advantages of flow cytometry?

Answer 38

1. Simultaneously detecting multiple fluorescent & physical characteristics of cells 2. Highly correlated data in a spreadsheet format 3. Fast detection rate with large sample size 4. With‘Gating’, useful information about a sub-population of cells can be obtained 5. Real-Time Data Visualization 6. With a sorting setup, useful cells, organelles and chromosomes can be sorted & collected 7. Powerful technique for different kinds of study,

Question 39

What are some disadvantages of flow cytometry?

Answer 39

* Cannot locate where a target protein/organelle within a cell. * Cannot measure cell morphology. * Cannot chase temporal response from the same cell. * Not compatible with cells in a solid tissue [e.g. work for a solid liver cancer??]