Cell based methods including flow cytometry

Question 1

what is cell based assay

Answer 1

is analysis based on cell culture method where live cells are grown and used as model to access biochemistry and physiology of love and dead cells.

Question 2

what are common used methods in immunology

Answer 2

-isolation of cells include flow cytometry, magnetic beads
-apoptosis: trypan blue to visualize dead cells for cell viability
-microscope: cell counting, Immunohistochemistry IHC, immunofluorescence IF
-characterization of lymphocytes specificity and function: flow cytometry

Question 3

what is a buffy coat

Answer 3

is a fraction of anticoagulated blood sample after centrifuge that contain white blood cells and platelets

Question 4

how is leukocyte isolated from blood sample

Answer 4

by centrifugation of blood sample that separate red blood cell on the bottom and white blood cells and platelets on the top

Question 5

what are the leukocytes types and their marker

Answer 5

• CD4+ T helper cells - CD3, CD4
• CD8+ T cytotoxic cells -CD3, CD8
• B cells : CD19
• NK cells- CD56
  -macrophage - CD14
  -dendritic cells
  -basophils

Question 6

what are two method of isolating cells with magnetic beads

Answer 6

• direct labeling: is when specific antibody is labeled directly with microbeads /magnetic beads. occurs in one step
• indirect labeling: first there is primary antibody that binds to the antigen cell surface and microbeads will bind to primary antibody. occurs in two steps

Question 7

what are different ways of indirect cells labelling

Answer 7

• antibody microbeads
  -streptadin microbeads
  -biotinylated microbeaads
  -fluorochrome conjugated antibody

Question 8

what is positive and negative isolation their pros and cons

Answer 8

positive isolation: is labeling of wanted/target cells
pros; maximize purity and reduce washing steps
cons; activate target cells

negative isolation: is labeling of unwanted cells
pros; easy removal of unwanted cells
cons; had to yield purity for small targeted population

Question 9

what is the separate technique of MACS cells

Answer 9

• for positive selection: targeted cells are labeled with MACs microbeads are are separated in MACs column where labeled cells are collected as negative fraction and they will elute in the column and unlabeled cells are retains as positive fraction
• for negative selection; un targeted cells are labeled with biotinylated antibody and anti-biotin microbeads, and un target cells are retained in the column as the targeted cells passes the column and are collected as enriched positive fraction

Question 10

what do we use to evaluate cell viability and cell counting

Answer 10

we can use trypan blue which stain dead cells and we will be able to visualize it

Question 11

what is IHC

Answer 11

is the use of antibody labeled as specific agent through antibody-antigen interactions

Question 12

how is IHC used or perform its function

Answer 12

here antibody is chemically attached to the enzyme that can covert colourless substrate into coloured reaction product. the coloured product can be visualized in light microscope

Question 13

what is IF

Answer 13

IF is used to visualize protein in the tissues using antibody that is conjugated to the fluorescent dye.

Question 14

explain direct and indirect of IF

Answer 14

direct: is when primary antibody is directly conjugated to fluorescent dye and

indirect is when secondary that is conjugated with fluorescent dye is attached to primary antibody binding to cell surface of antigen for visualization of protein in the tissue

Question 15

what colour is B cells and HLA-DR are visualized

Answer 15

B cells is red and HLA-DR is green

Question 16

Characterisation of lymphocyte specificity and function

Answer 16

cell growth/ proliferate

Question 17

what are different ways to evaluate cell growth

Answer 17

-H-thymidine
- cell trace staining : we use flow cytometry

Question 18

why is H-thymidine is incorporated into DNA when cells divide

Answer 18

because it has high level of cell division

Question 19

what does it mean to have high level of radioactivity

Answer 19

means they have high level of proliferation

Question 20

how do we analyze the lymphocyte characterization using flow cytometry

Answer 20

by staining cells with cell trace stain

Question 21

what is ELISPOT assay

Answer 21

-is a simple tool to measure T cells responses
it can be use to know how many cytokine secreted by T cells. and also how many antibody was secreted by B cells

Question 22

how do we analyze if T cells produce cytokines by using ELISPOT assay

Answer 22

• we first add antibody specific in binding cytokines
• activated T cells are added of different effector function
• some activated T cells will produce cytokines which will bind to the specific antibody
• and bound cytokines are revealed with secondary antibody that is conjugated to the enzyme
  is spotted by giving insoluble precipitate

Question 23

how can you cunt number of Tcells secreted cytokine

Answer 23

-first add same amount of T cells in the control wells and other wells
-add peptide in other wells
-patient T cells will respond to the peptide by secreting cytokines
- because we know how many T cells we have we can know how many T cells secreted cytokines

Question 24

how is ELISPOT used to know how many antibody was secreted by B cells

Answer 24

by using specific antigen coated on the surface and enzyme labeled antibody

Question 25

what is cytometry

Answer 25

refers to measurement of physical and chemical characteristics of the cells.

example: cell marker, cell size and shape, cytokines production

Question 26

what is flow cytometry

Answer 26

is when such measurements are make when cells are passing through measuring apparatus

Question 27

what is flow sorting

Answer 27

extends flow cytometry by using electrical or chemicals means

Question 28

what is the application of flow cytometry

Answer 28

cell counting, cell staining

Question 29

what does flow cytometry tell us about cells

Answer 29

• its granularity
  -its relative size
  allow investigation of cell structure and function

Question 30

How is a dot plot generated?

Answer 30

dead cells
variable cells
apoptotic cells: cells die when they achieve maximum cell division and can no longer reproduce

Question 31

what is fluorochrome

Answer 31

are component of molecule that absorb light and emit fluoroscence. they contain aromatic group

Question 32

what is the importance of labeling antibody with fluorochrome

Answer 32

this can be used to mark cell surface of antigen

Question 33

How is a dotplot generated btn PE and FITC

Answer 33

• positive PE generation and positive FITC generation
• negative generation marking dead cells
• double positive PE and FITC generation

Question 34

what does dotplot interpretation show us (see note slide 38)

Answer 34

it can show us how many percentage of cells present in different generation

Question 35

What is the viability status of marked cell populations?

Answer 35

-cells that are both negative for annexin V and 7AAD has no viability or apoptosis, means that PS translocation has not occurred and plasma membrane is still intact.

• cells that are annexin V positive and 7AAD negative means that they are in early apoptosis but cell membrane are still intact
• cells that are double positive for annexin V and 7AAD means cell death or last stage of apoptosis, the cell membrane is not intact

Question 36

what are important factor for flow cytometry

Answer 36

• compensation: is needed when we are using multiple fluorophores whose emission signal overlap
• unspecific binding: can be due to Fc receptor binding, where specific binding is for high affinity and saturated cells and unspecific binding is for low affinity and unsaturated
• isotype control: use antibody of the same isotype as marker of the interest but from different species
• autofluorescence: know that cell fluorescent even if there is no satining added, autofluoroscent come from normal cell component including riboflavin and flavoprotein
  -multiplex analysis: analyzing using gate or subgate

Question 37

application of flow cytometry

Answer 37

-allow cell separation of the interest from heterogenous cell population

Question 38

how do you Analysis secretion of cytokines by Cytokine Bead Array

Answer 38

• sample is added to capture beads mix
• labeled antibody that is specific to the molecule/ primary antibody are added
• then captured analyte are analyzed by using PE-conjugated antibody

Question 39

summary must know

Answer 39

• leukocytes can be isolated by density gradient centrifugation, magnetic beads (MACS, Dynabeads) and
  flow cytometric cell sorting.
• Microscopy can be used to count cells (trypan blue) and to assess phenotype and localization of cells in
  tissue sections by immunofluorescence and immunohistochemistry. Number of cells analysed fairly
  limited. Traditionally, few markers analysed. More extensive multiplexing only recently developed.
• Flow cytometry can be used for analysis of cells in suspension. Multi-parameter analysis is and has for
  long been common (up to 20-30 markers in conventional flow cytometry) and thousands of cells can be analysed per second. Gating strategies can be applied for detailed analysis of specific cell types. Spectralinstrument allows)
• Production of soluble mediators can be assessed with e.g. ELISPOT, Luminex and CBA. Intracellular cytokines can be assessed with flow cytometry.