BMS Exam 1 Flashcards

Histology

microscopic examination of tissues to examine structure and composition

immunohistochemistry

tagging proteins/tissues with fluorescent antibodies to visualize w/ fluorescence

in-situ hybridization

visualizing nucleic acids/ DNA or RNA chromosomes with fluorescence imaging

immunoprecipitation

precipitation of a specific protein out of solution via antibody isolation

5 steps in histology preparation

1. collection

2. fixation

3. embedding

4. sectioning

5. visualization

collection

surgical/ ultrasound

why can’t you use human tissue?

no true “control”, pathogen spread

fixation purpose

keeps structure from deteriorating after separation from organism: prevents lysis and blocks metabolism by denaturing certain molecules

physical fixation methods

microwaving, heating, cryopreservation

why cryopreservation

freeze dry tissue for diagnostic speciments

chemical fixation method

perfusion

perfusion

saturation of a tissue in a chemical that blocks metabolic processes

formaldehyde and formalin

creates protein crosslinks for stability but blocks antigen sites

factors that influence chemical fixation

temeperature, time, penetration rate, size/volume of tissue sample, pH and buffer

methods of embedding

paraffin, OCT, cryoblock

paraffin

stable for years, heated and poured over sample before cooling to room temp

cryoblock

when heat may damage tissue, for IHC and ISH

OCT

optimum cutting temperature, snap freeze tissues, most common form of embedding

sectioning instruments

microtome and cryostat

microtome

fine steel blade cuts paraffin- single layer of cells

cryostat

cooled to -20 C, fragile specimen so thicker sections

Hematoxylin and Eosin

basic: dyes nuclei blue
acidic: dyes cytoplasm pink

Cresyl violet

detection of ion levels (ferric ions) deposits

Luxol fast blue

stains myelin blue, neurons violet

Perl’s Prussian blue

stains neurons and glia blue/violet

Periodic acid- Schiff (PAS)

used to detect polysaccharides, glycoproteins, glycolipids

Oil red O

dyes fatty acids bright red

steps of immunprecipitation

1. protein treated with primary antibody

2. protein A and G attached to antibody, making protein insoluble

3. centrifuged to pellet protein

chromatin immunoprecipitation

preserves protein-DNA interactions (between transcription factors)

complex immunoprecipitation

preserves protein-protein interactions (after post-translational modifications- adding of groups onto protein)

ribonucleoprotein immunoprecipitation

preserves RNA-protein interactions: between post transcriptional modifications (poly A tail and 5’ cap)

primary antibody

antibody that directly attaches to protein of interest- determined by Fab region

secondary antibody

antibody that binds to and recognizes primary antibody, fluorescent tag

fluorochrome

fluorescent molecule that attaches to secondary antibody

direct immunofluorescence

primary antibody tagged with fluorochrome attaches directly to protein of interest

indirect immunofluorescence

secondary antibody w/ fluorescence tag binds to primary antibody (binded to target protein)

hydrodynamic focusing

cells flow into flow cytometer in single-file line into interrogation point (where laser meets cell), allows for single cell analysis

how do cells move in flow cytometer

bernoulli effect: laminar coaxial flow, pressure lowers as area narrows

why lasers?

coherent: single wavelength, no divergence

forward scatter

determines cell size; higher scatter = larger cell

side scatter

determines cell complexity/granularity (more organelles = higher granularity)

% positive

percent of cells that express protein of interest

mean fluorescent intensity

measure of fluorescence in antigen expression (average density of protein in cells)

how to read a bit plot

higher y = larger cell

higher x = more complex/granular

closer clustering = same cell type

how to read a fluorescence histogram

peak on left controls for auto-fluorescence

tighter peak = more homogenous

further to right = greater presence of antigen

gated peaks

isolate to determine which intensity belongs to cluster

function of dichroic and bandpass filters

filter the light from a wavelength to individual fluorochrome types

dichroic filters

some light deflected off of filter, some passes through- separates colors

bandpass filter

allows only specific wavelength through, blocking all others

Fluorescence- activated cell sorting (FACS)

cells are stained with different fluorochromes per cell types

droplet = individual cell, charged based on emission wavelength, directed into vessel based on opposing charges

advantages of flow cytometry

rapid analysis of many cells, greater accuracy, quantitative data, operator independent, not labor intensive

disadvantages of flow cytometry

costly, little information about individual cells, things can go wrong

ELISA

enzyme-linked immunosorbent assay: quantifies proteins in a solution

ELISA methods

antibody specific to cytokine bound to plate walls, sample added and binds to antibody. substrate added: color reacts based on protein concentration

ELISA purpose

quantification of protein but not how many cells produce protein

EliSPOT

quantification of cytokine-producing cells, not concentration of cytokine

toxicity assay

determines mechanism that cells die (necrosis or apoptosis)

propidium iodide

binds to DNA- detects necrotic cells (explode inside of cells), red

Annexin V

binds to cell membrane- only remains if apoptotic- green

mitochondrial activity assay

MTT dye injected, mitochondria breaks down into formazan, turns purple. Cells measured via optical density- brighter colored = greater mitochondria activity

analysis of cell cycle

cells fixed with DNA fluorochrome, analyzed w/ fluorescence histogram. larger fraction in G2 = rapid cell division

applications of cell cultures

toxicity assays, responses to stimuli, molecular interactions, microbial interactions

limitations of cell culture

what occurs in vitro does not always predict in vivo- complex interactions

culture hoods

maintain sterile working area to minimize contamination

horizontal laminar flow hood

airflow moves out toward researcher

HEPA filter

stops movement of particulate matter from air, moves out of area

BSC Class II hood

similar to horizontal laminar flow hood but also traps particulate matter from hood inside- lower risk of contamination

incubator

maintains optimal environment for cell growth and division, water jacket keeps temp stable

phase contrast microscope

inverted mirror- better visualization with darker spots + more detail of cells

centrifuge

spins suspension of cells in media to create pellet of cells at bottom

physics of centrifuge

centrifugal force pushes cells down, RCF is dependent on rotor size

centrifuge speeds

cells: 3000-4000g

DNA: 130000g

Nucleic acids: 200000g

nanoparticles: 60000g

freezers at 4C

store normal reagents and media

freezers at -20C

store DNA, viruses, reagents, enzymes

freezer at -80C

bacteria strains

cryopreservation (liquid nitrogen tanks)

storage of cells for extreme long term

components of mammalian cell culture medium (7 things)

1. amino acids

2. salts

3. vitamins

4. glucose, phenol red, antibiotics, growth factors and cytokines

function of amino acids in cell medium

building blocks for cells to divide and grow- brought in via diet

function of salts in cell culture

maintain osmolarity of cells

function of antibiotics in cell culture

prevent well contamination

potential sources of tissue for culture

• human biopsy: discarded tissue, cadavers

• animal tissue/embryos

steps of creation of primary culture from tissue

1. dissect tissue from source

2. cut down into smaller pieces

3. enzyme digestion

4. cells placed in culture vessel

5. provided with culture to grow

passage of cell cultures

expanding population from initial culture

confluence

minimum # of passages a cell can take before growth levels off

contact inhibition

cells stop growing when they come into contact with each other

caveat of cell passage

may not be the same as original population

Adherent cell cultures

attach to wall of cell culture wells

passage of adherent cell cultures

1. aspirate culture media

2. rinse with buffer

3. detach cells from well with trypsin/EDTA

4. dilute cells from culture

5. transfer into new wells

non-adherent cultures

do not stick to culture wells, lymphocytes and transformed lines

steps of non-adherent culture

proliferate in suspension, density determined by turbidity (cloudiness) or cell count, dilution and transfer

cryopreservation methods

store in liquid nitrogen flask
10% DMSO + cell media: prevent crystals from forming in media

characteristics of cells

markers (protein, enzyme)

adherence

sensitivity to injury

density

positive selection

isolating specific protein

negative selection

getting rid of other cells types

fluorescent tagging

antibody fluoresce when attached to cell marker of interest

centrifugation

separates cells by density within given separation

magnetic cell sepration

secondary antibody has metal bead: detected by magnetic column to separate cells

cloning

replication of identical cells from single cells

process of cell counting by hemacytometer

cell culture placed on machine with grid

number of cells counted within each square

outside + = large cells, low density

inside + = small cells, high density

trypan blue

differentiates dead vs alive cells: absorbed by dead cells, not live