Cell Culture Techniques

Explain Primary cells, Cell lines and Stem Cells

Primary Cells- isolated tissue cells used for experiments

Cell Lines-immortalized cells that proliferate under right conditions

Stem Cells- self renewable, specialized cells

Hayflick limit

Explians mechanisms behind cellular aging

Telomeres

End of chromosomes that consist of a short repeating DNA sequence

Telomerase

- endogenus ribonucleoprotein (protein + RNA)

- stabilizes and elongates telomers

- Protein -> hTert (human telomerase reverse transcripzase)

- RNA subunits -> TERC/TR/TER

- expressed in embryonic stem cells

From where do we getr cell lines?

- derived from human cancer cells

- derived from healthy somatic cells (building blocks of the body)

Types of Stem Cells

totipotent, pluripotent, multipotent

Why are stem cells important?

They can differentiate into into any specialized cell

What are feeder Cells?

fibroblasts that secrete factors to prevent stem cell differentiation

What chemicals support stem cell pluripotency?

Growth factors

Different ways to culture cells

1) monolayer cell culture

2) 3D cell culture:a) scafold free b) scafold based c) hybrids

Labeling of Cells: living vs fixed

- living have an intact membrane, labeled with fluorescent proteins, DNA dyes, Cell structure probes and Dye labeled antibodies

- Fixed cells membranes are permabilized so you need a DNA probe for labeling

Fixation of Cells (permabilization)

1) MeOH/EtOH (cells are dried out, membranes are strongly affected)

2) PFA (paraformaldehyde, sometimes issues with antibody binding, fluorescence gets destroyes, strctures are conserved by cosslinking)

What ypes of antibodies detection tools are used?

Direct -> antibody goes directly to the antigen (better for detecting lower concentrations of ag)

Indirect -> 1st antibody is detected with 2nd (better for detecting higher concentrations of antigens)

What are fluorophores?

Chemical compounds that absorb/emit light of specific wavelengths; can be a dye or protein

Methods using antibody detection

Immunobloting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, microscopy, immunopercipitation

Problems with Brightfields microscopy

living cells are full of water makig it harder for them to absorbe and the light passes through them almost unaffected

Where is phase-contrast microscopy (PCM) used and why?

Solution for the brightfield microscopy problem,

When light passes thorugh the dense medium or part dense part of the cell there, the light decrease in velocity and decrease in amplitude or intensity. Leading to destructive interference.

Fluorescence microscopy: Artefacts

- unspecific binding to ABs create interesting spots and blubs, this creates false positive signals that look like real protein locations but aren't

- Low signal/ noise ratio. Noise is the unspecific binding. When high noise and low signal its hard to distinguish the real target from the background, leading to wrong results.

Fluorescence microscopy: Controls

- test concentration of AB to reduce background

- use 2nd antibody

- compare to cells with KO of your protien of interest

- use strains for cellular compartments or structure if relevant

What happenes in light scattering principles and whats important there?

Simlar to FACS just once the laser goes through the cell it scatters the laser light in all directions, leading the fluorescent molecule inside ot the cell to get excited causing emmision.

Forward Scatter (FSCH): small shallow angle light collected by th lense and measured by PMT (photomultiplier tube). This measures the size.

Side Scatter (SSCH): large wide angles that measures the granularity.

Where is gating used?

Light scattering principles

What do we use for Cell Viability?

Trypan blue

What do we use for CYTOTOXICITY?

Resazurin because of mitochondrial enzymes will reduce it

What do we use for Proliferation detection?

EdU- thymidine that can go into DNA during ploriferation as they need thymidine to form building blocks

Types of cell death?

Apoptosis- programed

Necrosis- cellular injury

What do you use for detecting early and late apoptotic/necrotic cells?

Fluorescent probes:

-Anexin V (binds to phosphatidylserine PS which is found in the inner side of the membrane, this is labeled with a fluorophore that is exposed only in apoptotic fase FLIP-FLOP)

-Propidium Iodine (used to label the nucleuse)

What do you use to detect only late apoptotic fragments of DNA?

TUNEL Assay- make cell do apoptosis, fixation with PFA, Incubate with TdT and biotine 11 sUTP, detection with fluorescent label, staining of DNA, analysis by FACS

Types of gene delivery in the mamalian cells?

Chemical (calcium phosphate percipitation, DEAE-dexran method, liposome transfection)

Non-Chemical (electroporation)

Particel Based (particle bombardment, magnetic particle transfection)

Virus Mediated (viral transduction)

Calcium phosphate percipitation explain?

Bind a DNA of interest with CaCl2 and add a 2nd solution of HEPA buffer that contains a phosphate. Ca 2+ and PO4 3- will combine to form a postivie charged calcium phosphate. This allows the complex with the gene of interest enetr the neg. membrane and allowing it to ercipitate in a cell.

Liposomal mediate transfection explained?

Use lipids to for a complex with the DNA. This complex is added to the cell where the lipid part will fuse with the cell membrane resulting to a release of DNAinto the cytoplasm.

What is Electroporation and Electroporation next gene?

Non-chemical gene delivery.

Plasmid enters the cell by applying a high votage creating pores in the membrane.

With the next gene its the same principle just the plasmid goes directly to the nucleus.

Which is one non chemical method that can also be used? (except electroporation)

microinjection and gene gun

Gene Gun

Usually used for plant cells but can be used for mamalian.

DNA is coated into tiny heavy particles. This DNA is loaded in a plastic disk where high pressure is used. He pressure builds up and until the plastic disk breaks and makes the particle accelerate in high speed leading to the DNA penetrating the cell wall and membrane if the target plant.

Reporter proteins vs Tags

Tags.. short artifitial peptide sequences detected with specific antibodies

Reporter Protein.. larger portein from non mamalian species with enzymatic activity.

- Short tags less likely to interfere with protein

- Reporter protien easier to detect

- Tag easier to insert

How does a reporter look in a plasmid?

promoter + reporter

promoter + reporter + 3'UTR

How to we separate 2 functional proteins after synthesis?

IRES and 2A

How does the visualization work with the GFP?

GOI will fuse with the GFP and get synthesized with the GFP which allows detection.

What are potential pitfalls of GFP with GOI?

overexpression, GFP may stops folding, signal too weak, spectral overlapp

What do you do as troubleshooting if the GFP GOI doesn't work?

Try N or C terminal fusion if one is not working

Typs of viral expression

Transistent (plasmid is delivered in the cell but no in the genom)

Stable (plasmid is delivered into the cell genetic material)

Name the Yamanaka factors

Oct4, Sox3, KLF4, c-Myc

What do you need for a KD?

RNAi (siRNA, shRNA and miRNA)

Where did CRISPR come from?

It was first found in bacteria as they use it as an immune respons

What is the difference with CRISPR/Cas9 in bacteria and lab?

In the lab we use sgRNA while in bacteria we have gRNA(pre crRNA + tracrRNA)

CRISPR/Cas subtypes

Cas 9, Cas12, Cas13

What are the different possibilities to performe a KO?

1) Creating a random idel which causes a perminant STOP codon

2) Deleting 1 or more exons

3) Exon intron fusion

What are the 2 options of a gene KI?

NHEJ- non homologus end joining

HDR- homology direct repair

What does the Cas9 sequence recognize to attach the sgRNA to the gene of interest?

PAM sequence containing any sequence + 2 guanin sequences

What does the HDR need to repair the cut gene?

It need a template that contains homology arms which match the DNA sequence on the side of the cut perfectly. This tricks the cell to think the piece of DNA belongs there

How can you control the life spam of a protein?

Using dTag because it will create a bridge of the protein to an E3 ligase for ubiquitination. Ubiquitine on the protein will be recognized by the proteasome for degradatino.

What is dCas9?

Nuclease deficit or dead Cas9 that doesn't cut the DNA

What cargo do you use for CRISPRi

KRAB

What is the affect of CRISPRa

Calls RNA polymeraze to the gene of interest.

What is NanoLuc and for what method is it used?

NanoLuc is an enzyme used for DNA labeling.

Method is called DNA looping where the NanoLuc + bioluminescence will release a color which you can track on a microscop.

What are the 2 types of CRPSPR screen?

arrayed screen

pooled screen

What other method isntead of DNA looping do we have for DNA labeling?

Chromatin imaging

Rank the potenncy of stem cells

1) totipotent -- can form everything

2) plutipotent -- can form everything except placenta

3) multipotent -- cells in a specific tissue, forms organs

What are organoids?

3D structures that mimic organs

What is the porblem of organoids and how do we solve it?

When they get bigger the cell center often dyes. This happenes due to low blood supply. Can be fixed by developing organoids on a chip system so they pump tiny chanells to deliver oxygen and nutrients deep into the tissue.