Preparation of peripheral blood lymphocytes

Question 1

What does heparin do?

Answer 1

prevents blood clotting before isolation of leukocytes

Question 2

What is the best way to isolate a leukocyte population?

Answer 2

FACS and FCM

Question 3

What are some disadvantages of FACS

Answer 3

• cost
• availability of equipment
• may need multiple Abs
• markers may not be on a cell type (e.g. no sIg on plasma cells)
• availability and quality of Ab.s

Question 4

What is a tetramer, what does is look for, why does it need to be tetrameric?

Answer 4

MHC heavy chain + B2M + fluorochrome + specific peptide + biotin + streptavidin

it looks for a specific T cell via FACS or fluorescence microscopy

Needs to be a tetramer because the TCR-MHC interaction is really weak

Question 5

What are the major CD Ags on granulocytes, monocytes, Th cells, Tc cells, an activated T-lymphocyte, a B lymphocyte, and a thrombocyte?

Answer 5

Granulocyte = CD45 and CD15
Monocyte = CD45 and CD14
Th cell = CD45, CD3, and CD4
Tc cell = CD45, CD3, and CD8
activated T-lymphocyte = CD45, CD3, and CD25
B cell = CD45 and CD19
Thrombocyte = CD45 and CD61

Question 6

Describe panning/magnetic bead cell sorting (MACS)

Answer 6

Have a magnetic bead on an Ab. probe –> incubate Ab with cells –> run the cells through an external magnetic field –> those with the Ab will remain in the column and those without will elute

Question 7

How can you deplete non-target cells?

Answer 7

Use a specific Ab. to the cell type you want to deplete –> add complements –> complement mediated lysis of non-target cells

Question 8

what is density?

Answer 8

mass per unit volume of a substance, often expressed in g/mL

Question 9

what is differential centrifugation?

Answer 9

separates particles on the basis of their size by a series of centrifugations at different speeds and times

Question 10

What is density gradient centrifugation?

Answer 10

separation performed in a supporting column of solution in which the density and solution concentration increases towards to bottom of the tube

Question 11

What is discontinuous/step gradient?

Answer 11

composed of layers with abrupt changes in density and/or concentration from one layer to the next

Question 12

Describe how we can use a discontinuous/step gradient to isolate leukocytes

Answer 12

Add histopaque to a centrifuge tube –> add unfractionated blood onto the histopaque –> centrifuge –> top to bottom: serum - buffy coat - histopaque - granulocytes - RBCs –> remove buffy coat

Question 13

Which cells are in the buffy coat?

Answer 13

monocytes and lymphocytes

Question 14

what is a cytospin?

Answer 14

instrument used to collect a solution for display on a slide (puts all the cells in one place for better visualization)

Question 15

What are some important centrifuge basics (i.e what should you do or not do when using a centrifuge to make sure you do not destroy the entire lab)?

Answer 15

• make sure you’re using the correct rotor for the centrifuge
• never exceed the max g force for the rotor
• make sure you’re using the correct centrifuge tube for the job (tubes may break if centrifuged at too high of a speed and plastic tubes can break if not in appropriate adaptors)
• pre-cool centrifuge and rotors for high speeds
• never walk away from a centrifuge until it reaches top speed (ensures it is balanced)
• always balance opposing tubes