5 differentiation assays, PCR, flow

differentiation assay

experiment inducing stem cells to become specialized cell types

adipogenic differentiation

process where stem cells become fat cells

osteogenic differentiation

process where stem cells become bone cells

oil red o stain

dye used to detect lipid droplets in adipocytes

alizarin red stain

dye used to detect calcium deposits in osteoblasts

PCR

technique used to amplify specific DNA sequences exponentially

DNA template

contains target sequence

usually genomic DNA, plasmid DNA

dNTPs

nucleotide building blocks used by DNA polymerase during DNA synthesis

primer

short DNA sequence that initiates DNA synthesis in PCR

forward + reverse

DNA polymerase

enzyme that synthesizes DNA from nucleotides

common: Taq

thermostable, fidelity, speed, specificity

thermostable polymerase

polymerase capable of functioning at high temperatures (ex. Taq)

PCR buffer

pH and ionic conditions

MgCl2

stabilize interaction b/w polymerase and DNA template

RT-PCR

convert RNA into cDNA before PCR amp

measure gene expression

reverse transcriptase

enzyme that synthesizes DNA from RNA template

RT vs. normal PCR

reaction does not cycle temp, each RNA molecule is converted to ONE cDNA molecule, no amp occurs during RT, amp occurs during subsequent PCR step

qPCR

measuring DNA amplification in real time during each cycle of the reaction

after each cycle, fluorescence intensity is measured (proportional to amount of PCR product, increases as DNA accumulates)

Ct value

PCR cycle number where fluorescence crosses the detection threshold

low = high starting DNA

high = low starting

Baseline phase

early cycles, little signal

exponential phase

PCR stage where DNA amplification is most accurate for quantification

plateau

reagents limited

relative quantification

comparing gene expression levels between samples

controls for diff in RNA amount

final result: ratio of target gene expression relative to control

absolute quantification

determining exact number of DNA/DNA molec

create standard cube using samples w/ known concentrations then compare experimental Ct values to it

ΔCt method

calculation comparing Ct of gene of interest with housekeeping gene

ΔΔCt method

calculation comparing ΔCt values between experimental and control samples

flow cytometry

technique measuring physical and chemical properties of individual cells in suspension

event

single cell measurement recorded by the cytometer

Ab-fluorophore

fluorescent molecule used to label specific cellular components

absorb and emit light at specific wavelengths

fluidics system

aligning cells into single file flow through the laser

optics system

lasers and filters used to excite fluorophores and collect emitted light

electronics system

converts optical signals into digital data

forward scatter (FSC)

light scattered at small angles indicating cell size

side scatter (SSC)

light scatted at 90 degree indicating cell granularity or internal complexity

ex. lymphocytes are low

CD marker (cluster of differentiation)

surface protein used to identify immune cell populations

compensation

mathematical correction removing fluorescence spillover between detectors

without, false double-positive populations may appear

spectral flow cytometry

capturing full emission spectra to distinguish many fluorophores

pros: 20-40+ markers simultaneously, $$

conventional flow

filters to send fluorescence to specific detectors

mass cytometry

uses heavy metal-tagged Ab detected by mass spectrometry

FACS

fluorescent activated cell sorting

flow cytometry method that physically sorts cells based on phenotype, electrically charges them and uses plates to deflect/sort them

flow cytometry experiment design

planning markers, fluorophores, controls, acquisition parameters

unstained control

sample without fluorophore used to measure background autofluorescence s

single colour control

one fluorophore to calculate compensation for spectral overlap

fluorescence minus one control

all but one

determine gating boundaries for that marker

gating

remove debris/select general population

remove doublets (to prevent incorrect counts/sorting error)

gate live cells

gate population if interest

Fc receptors

receptors on immune cells that bind Ab Fc regions

can cause non-specific Ab binding = false positives

so use blocker to prevent this

immunomagnetic separation

method using Ab-covered magnetic beads to isolate cell types

faster than FACS

primary antigen

highly expressed marker

secondary antigen

moderately expressed marker

tertiary antigen

low expressing marker

antigen density

low antigen density/dim marker = bright fluorophore

high/bright marker = dim fluorophore