LDH Assay

Question 1

Apoptosis

Answer 1

NF-kB mediated

Cell suicide

Question 2

Pyroptosis

Answer 2

Caspase-1 mediated

Inflammatory molecules released, affects neighbouring cells

Question 3

CSF

Answer 3

critical cytokine colony stimulating factor

Question 4

PAM3CSK4

Answer 4

Lipoprotein
TLR 1/2 ligand
mimics proinflamm bac lipoproteins
A1, A3, A4, A5

Question 5

LPS

Answer 5

Proinflammatory molecule
Activates TLR4
Endotoxin
Can set off TSS
B1, B3, B4, B5

Question 6

Alum

Answer 6

Adjuvant
Activated BMDM in conjunction with LPS
B2, B3, B4, B5

Question 7

Alum

Answer 7

Adjuvant
Activated BMDM in conjunction with LPS
B2, B3, B4, B5

Question 8

BAY 11-7082

Answer 8

Inhibits NF-kB/apoptosis

A5, B5

Question 9

Z-WEHD

Answer 9

Inhibits caspase-1/pyroptosis

A4, B4

Question 10

Nigericin

Answer 10

A2, A3, A4, A5

Question 11

Triton-X100

Answer 11

Complete cell death

Question 12

Positive Control

Answer 12

Complete cell death,
Triton X-100
A6

Question 13

Negative control

Answer 13

B6

Untreated BMDM

Question 14

LDH solution

Answer 14

Lactate, Iodonitrotetrazolium chloride, and NAD+/Diaphorase

Question 15

Reading

Answer 15

Od 492nm

Question 16

Reading

Answer 16

Od 492nm

Question 17

3H-thymidine incorporation

Answer 17

Add radioactive thymidine which will be incorporated into dividing cells

Question 18

PBMC

Answer 18

Peripheral Blood Mononuclear Cells
Used to measure activation in lymphocytes and T cells
Add Ag –>culture –>Add 3H-Thy –> measure DNA and how much 3H-Thy is present
cpm (counts/min) increased w activation

Question 19

MTT assay

Answer 19

IL-2 assay
Dye added to cells, cleaved by enz in cells
Growing cells cleave more dye
IL2 containing supernatants added to IL2 dependent cell line
IL2 dependent cell line then proliferates
Can measure if IL2 is functional

Question 20

IL2 ELISA

Answer 20

measures amt IL2 prot by capturing it

Question 21

IL2 Staining by Flow Cytometry

Answer 21

advantage: per cell basis
Can ID which cell is making prot
Brefeldin A- maximize the measurement of cytokines inside the cell by preventing secretion
Stain T cells stimulated with antigen for the production of IL-2
Activated T cells are permeabilized with detergent and stained with fluorescently-labeled antibodies against IL-2.
Can use fluorescently-labeled antibodies specific for cell surface markers which allows direct identification of the cells producing IL-2.

Question 22

LacZ T cell Activation Assay

Answer 22

Promoter for IL2 has LacZ gene
Req T cell hybridomas
B-galactosidase is indirect measurement of Tcell activation
Add substrate chlorophenol red galactoside
(CPRG) –> when hydrolyzed by b-gal it changes from yellow to red/purple.