purification of lymphocytes

Question 1

Describe the layers (& %) seen after blood has been centrifuged. What is a buffy coat?

Answer 1

• Plasma (55%)
• Buffy coat (= WBC) & platelets (<1%)
• erythrocytes (45%)

Question 2

*How does density gradient centrifugation improve separation of WBCs?

Answer 2

To obtain pure sample of buffy coat need to lyse RBC w/ water => then put isotionic saline (WBC resistant to water)*

Question 3

Describe the layers and what cells are found in each layer after centrifugation using Ficoll Hypaque?

Answer 3

• Yellow: plasma, platelets
• precipitate: mononuclear/agranular WBC
• colourless: Ficoll-hypaque
• purple: RBC & granulocytes

Question 4

Describe the magnetic bead separation technique.

Answer 4

1. small magenetic beads coated w/ monoclonal AB
2. added to blood => incubated
3. specific cell binds to Ab on bead = form rosettes
4. magnet placed near tube = attract beads w/ rosettes
5. other cells & serum removed

Question 5

What information does forward and side scatter give in flowcytometry?

Answer 5

• FWD scatter: size

- Side scatter: granularity (complexity of cytoplasm)

Question 6

Describe the components of a flow cytometer.

Answer 6

• Fluidics: cells flow in single file
• Optics: generate & collect light
• Electronics: process optic signals

Question 7

How are cell sorted with a Fluorescent Activated Cell Sorting (FACS)?

Answer 7

1. Ab conjugates (flurorescent dyes) react w/ cells
2. cells pass through flow cytometer & labelled cells are detected by lasers
3. cells become charged => deflected from by the electric field
4. cells sorted

Question 8

List the steps in the serology based HLA typing technique.

Answer 8

1. serum containing known specific anti-HLA Ab in the wells of tray
2. lymphocytes are added to serum. Rxn occur if lymphocyte has specific HLA type - Ab can bind to
3. Rabbit complement is added to activate complement via classical pathway => enlarge
4. detect killed cells wby staining w/ ethridium bromide (eosin) [live cells smaller & refractile]

Question 9

What are the various ways you can do with neutrophil functional assays.

Answer 9

• Phagocytosis: to measure uptake of bacteria (labelled)
• Intracellular killing: known concentration of bacteria is reacted w/ neutrophil => plate out & colony count = how many killed
• directional migration: see if they can move through filters or gradients twd chemoattractant
• Measure up-regulation of surface markers using monoclonal Ab w/ flow cytometry = see if they express Ag on surface

Question 10

Describe the cytotoxic T cell functional assay.

Answer 10

measure cytotoxic T cell ability to kill a target cell (express K562) - labeled w/ radio isotope (51 CR)- by measuring the release of 51Cr in surrounding media

Question 11

What is HLA?

Answer 11

Human leukocyte antigens (MHC antigens)

Question 12

MOnoclonal Ab of NK cells

Answer 12

CD36 or CD16