LECTURE 3

Question 1

2 ways to process tissue for microscopy

Answer 1

freezing and formalin fixing

Question 2

what does formalin fixing do?

Answer 2

fixes protein conformation

Question 3

5 examples of stains and what they stain

Answer 3

hematoxylin –> nucleus
eosin –> proteins
hemosiderin –> Fe
alcian blue –> mucin
silver nitrite –> nerves

Question 4

monoclonal vs polyclonal ab

Answer 4

monoclonal = ab with 1 epitopes
polyclonal = ab with many epitopes of 1 protein

Question 5

why are monoclonal ab helpful?

Answer 5

more specific and can optimize affinity

Question 6

how are monoclonal ab made?

Answer 6

1. inject Ag into animal
2. activated plasma cells from spleen fused w tumour cells in PEG
3. HAT screens for non-fused cells
4. screened for 1 specificity

Question 7

how are polyclonal ab made?

Answer 7

1. inject Ag into animal
2. activated plasma cells make Ab

Question 8

2 ways the secondary Ab amplifies signal

Answer 8

1. fluorescent marker
2. enzyme

Question 9

benefit of fluorescent marker on secondary Ab

Answer 9

more sensitive if frozen tissue

Question 10

describe the nomenclature for mouse-anti-rat-il2

Answer 10

mouse immunized with rat IL2

Question 11

describe IHC

Answer 11

stains proteins on tissue section and uses light microscopy

secondary Ab linked to enzyme, substrate is added to give specific combo and and counter stained w hematoxylin

Question 12

IHC for frozen tissue vs FFPE tissue

Answer 12

frozen is best, Ag are not fixed

FFPE may require Ag retrieval via enzyme treatment or high pressure boiling

Question 13

describe confocal microscopy

Answer 13

stain w many diff Ab and image many layers of cell/tissue

also allows spatial filtering techniques to eliminate out of focus light or glare if too thick

Question 14

describe ELISA

Answer 14

• primary Ab bound to plastic plate (to CAPTURE)
• secondary Ab bound to captured Ag and linked to biotin (to DETECT)
• streptavidin-HRP added which binds biotin
• substrate added to induce colour change proportional to [Ag]

must make standard curve

Question 15

describe WB

Answer 15

SDS-PAGE makes proteins negatively charged and separate by size to transfer to membrane and then visualize

Question 16

purpose of flow cytometry/FACS

Answer 16

analyze/separate cells, organisms, and particles as they pass thru lasers

Question 17

microscope for flow cytometry

Answer 17

high speed automated fluorescent microscope, uses photo detectors instead of eyepiece/camera

Question 18

compensation in flow cytometry

Answer 18

if spectra overlap, must compensate otherwise will give false double positive

Question 19

forward scatter?

Answer 19

granularity

Question 20

side scatter?

Answer 20

size

Question 21

purpose of PCR

Answer 21

amplify specific DNA sequence using primers for GOI which bind denatured DNA

Question 22

next generation sequencing and 4 steps

Answer 22

cheap sequencing by a machine!

1. extract DNA
2. make DNA library
3. sequencing
4. analysis

Question 23

3 types of NGS

Answer 23

1. genome sequencing (most expensive, low accuracy, low # reads)
2. exome sequencing (a bit cheaper, more accurate, higher # reads)
3. targeted gene panel (cheapest, most accurate, most reads)

Question 24

purpose of qPCR

Answer 24

measure specific transcripts/gene expression using cDNA made from RNA via RT

Question 25

describe qPCR graph

Answer 25

increasing when fluorescence > background

threshold cycle (CT) = a bit higher than 0, where fluorescence becomes measurable

Question 26

standard curve for qPCR

Answer 26

log [ ] vs CT

Question 27

how can you calculate [cDNA] of unknown sample?

Answer 27

using its CT value`

Question 28

purpose of microarrays?

Answer 28

detect 1000s of genes at once

Question 29

4 steps of microarrays

Answer 29

1. extract RNA from experimental and control samples
2. RT to make cDNA labelled with diff colour probe
3. mix samples
4. add to microarray slide with 1000s of wells of known DNA sequence/gene
5. measure gene expression

Question 30

when is microarray red?

Answer 30

if experimental expression > control

Question 31

when is microarray green?

Answer 31

if experimental expression < control

Question 32

3 critical aspects of tissue culture

Answer 32

1. sterility
2. temp
3. O2/CO2

Question 33

describe colour of media for tissue culutre

Answer 33

yellow = acidic = cells are growing

Question 34

what are transwells?

Answer 34

to separate cell populations, can see effect of soluble factors btwn cell types

Question 35

why do we look at metabolism of cells?

Answer 35

cancer cells have diff metaoblic pathways than healthy cells, immune cells have diff metabolism when active

Question 36

2 quantities we can look at when studying metabolism

Answer 36

1. O2 Consumption Rate –> [O2] over time, based on mt respiration/ox phos
2. Extracellular Acidification Rate –> driven by lactate from glycolysis

Question 37

purpose of glycolytic stress test

Answer 37

what happens to cells with increased glycolysis

Question 38

describe glycolytic stress test

Answer 38

use OLIGOMYCIN which inhibits ATP synthase so no ATP from ox phos
- increased glycolytic capacity = increased ECAR

then block oligomycin with 2-DG
- decreased glycolytic capacity = decreased ECAR

the increase induced by oligomycin = glycolytic reserve

Question 39

describe mt stress test

Answer 39

1. use oligomycin to decrease OCR
2. use FCCP to cause H+ leak and increase OCR to max. respiration
3. use ROTENONE/ANTIMYCIN A to inhibit ETC and decrease OCR

Question 40

what does an increase in ECAR mean?

Answer 40

increased cell activation, proliferation (i.e. metabolism)

Question 41

what does a decrease in OCR mean?

Answer 41

increased metabolism