ALAT Exam 1 Flashcards
Principles of Flow Cytometry
The fluorochrome gets excited by the laser pushing it into an unstable valance, will then begin to lose energy and produce light, you will always be reading the light at a lower energy than what the laser emits
Antibodies with fluorochromes attached to cell surface markers of interest
Emission
Wavelength given off by cell
Excitation
Wavelength given off by laser
Flow Cytometry
Forward scatter/side scatter are proportional to…
cell volume based on cell size, strucure and complexity, and nuclear lobularity
FSC measures…
Cell size
SSC measures…
cell granularity or internal complexity
How is DNA content measured through Flow Cytometry
Measured through the amount of propidium iodine or BRDU that is taken up by the cell and is emitted off when using flow cytometry
Cells are fixed and permeabilized, then stained with PI
How does PI measure DNA content?
irreversibly binds major and minor grooves of DNA
How is BrdU utilized in flow cytometry
BrdU is incorporated into dividing cells during S-phase
BrdU must be taken up into the cell through the cell cycle, this is great for cancer cells because they divide quickly, but moves slower than PI which permeabilize the cell membrane
Used to measure DNA content
What are the general steps for using BrdU in flow cytometry?
- Cells are exposed to BrdU for a fixed period of time (add to tissue culture plate/inject into animal)
- Collect, fix, and permeabilize cells
- Stain with anti-BrdU antibody
- Flow
Wait indicated time to allow for BrdU to be incorporated during cell division (Will depend on cell type and system being used)
Key steps regarding retroviral preparation
-
Day 1: Night before plate 293T cells, 4x106
This ensures that the plate is ~70% confluent, no debris should be present in the media so it does not get caught in the flow filter -
Day 2: Transfection
cotransfected b/c more than one plasmid
Envelope/packaging
TALL
Empty vector for cloning
4 hours after transfection change media
Lentivirus - can leave media until collection day
Retrovirus - MUST refresh media 24 hours before collection -
Day 4: Collection Day
Filter with 45 um filter
Freeze supernatant
What is the purpose of viral titering?
ensures use of the same amount of virus in each experiment
Guesstimation of viral units/mL
Steps utilizing Donor Mice in Bone Marrow reconsitiution
Donor mouse: euthanized mice that have their bone marrow stem cells flushed out with PBS
* Culture top notch bone marrow overnight with SCF, IL3, and IL6
* Populates overnight to ensure that potential cells can populate
* Cells are spun down and washed with PBS to inject into the tail of the mouse
* When performing flow cytometry on them green should be seen to ensure that cellular infection has occurred
The media can not be injected into the mouse only with PBS
Acceptor Mice in Bone Marrow reconsitiution
These mice are myeloablative through the use of busulfan injections in the tail (this takes the place of whole body irradiation) which creates space in the mouse for the donor stem cells to come in and home to the area
They are able to take the stem cells because all of these mice are genetically identical
How can you “turn off” tumors in Bone Marrow Reconstiution?
The system is under control of a tetracycline operator, meaning that the tumors can be turned on or off with the use of doxycycline
Why would you not want bufulsan to enterly wipe out the acceptor mouse’s stem cells?
Do not want this to be 100% lethal because when injected only T cells would be made, notch increases T cells, but no B cells or RBC would be made, making mouse anemic
Allows for better time course for mouse because they can stay healthy longer
Purpose of Busulfan Injections in Bone Marrow reconstitution
Takes place of whole body radiation
Causes myeloablation - destroys stem cells and leaves room for new viral BM
Purpose of 5 - FU in Bone Marrow reconstitution
Enhances for specific population of stem cells by killing the rest
Ensures strong hematopoietic cells
Transduction
Introduction of foreign DNA into a cell
Transfection
introduction of Plasmid (“naked DNA or RNA”) into a cell by lipid, chemical, or physical means
What are some ways to transfect?
Lipofectamine
Fugene
Jet-prime
Turbofect
Electroporation
Heat (bacteria)
TAT protein - from HIV - attached to DNA - facilitates transmembrane transport
Infection
Introdution of DNA or RNA into a cell by virus
Retroviral Infection
integration into genome
dividing (cycling) cells
viral recombination
HIV, HPV, HSV, EBV
Flu and COVID are retroviruses that do not integrate
Lentiviral Infection
Integration into genome
cycling or quiescent cells
Adenoviral Infection
No integration, temporary expression, requires boost
transient, in vacuole not nucleus
Ecotropic
mouse and rat host
Amphotropic
most mammals, including human hosts
Modification of retroviruses
The virus is cut into three parts, each having a LTR (long terminal repeat on each end); envelope, packaging, and transfer plasmid
* Allows virus to enter cells in culture but make it extremely hard for recombination to occur and for the virus to become contagious
* There is no replicating part so once the virus is in the cell it will not continue to replicate
* The more elements that are on different plasmids the less risk to the population
Virus production: Retroviruses
Done through transfection
Thaw competent cells and put on ice
Aliquot cells and DNA into snap cap tubes, 1 tube/ plasmid
Combine and heat shock - so DNA can enter the bacterial cells
Plate bacteria onto LB agar + amp plates and allow to incubate until colonies form
Place single colony into LB broth overnight
Mini-Prep to collect the DNA and spec for amounts
Combine DNA with serum free media
Incubate cells with turbofect for 20 min
Drop by drop add viral media to plate that was plated the night before with 4X106 cells
Can analyze transfection rate using flow after 24-48 hours
Do not want growth serum because you don’t want to kill cells or form toxic components
IRES
Internal Ribosomal Entry Sequence
Allows for the creation of two separate proteins because the ribosomes can bind in the middle of the sequence, without these the two proteins would be fused together
What is the purpose of constructing expression vectors?
Enable the expression of a specific gene or genes in a host organism, such as bacteria, yeast, or mammalian cells.
Know how expression vectors are constructed
Creating an expression vector involves inserting a specific gene into a plasmid vector.
This gene is then cloned into the vector using restriction enzymes and ligase.
The newly formed plasmid is then introduced into a host organism (typically bacteria), for replication and protein expression
Design (vector, PCR primers, etc.), PCR, Electrophoresis, Restriction Digest (specific enzymes to cut section you want to insert vector), gel purification, transformation, mini prep, sequencing
10 Hallmarks of Cancer
- Self-sufficiency in growth signals
- Insensitivity to antigrowth signals
- Apoptosis Evasion
- Limitless (immortal) replicative potential
- Sustained angiogenesis
- Tissue invasion and metastasis
- Deregulating cellular energetics
- Genome instability and mutation
- Avoiding immune destruction
- Tumor - promoting inflammation
Self-sufficiency in growth signals
Normal cells require growth cells to proliferate, but cancer cells escape this requirement
Ligand independent growth
Cycle faster → skip a lot of cycle checkpoints
Insensitivity to antigrowth signals
Normal tissues are protected from overproliferation by a variety of inhibitory signals, but cancer cells are insensitive of these
Ignores signals that inhibit cell growth
Apoptosis Evasion
Apoptosis is used by normal cells to prevent damaged or defective cells from continuing to divide; apoptosis is inhibited or disrupted in cancer cells.
Limitless (immortal) replicative potential
Normal cells have limited replicative potential due to telomere loss; cancer cells contain active telomerase (or other mechanisms) to maintain telomeres
Some cell lines have this property to be able to continue a cell culture
Sustained angiogenesis
(Solid) Tumor cells cannot grow beyond a few mm without a blood supply; cancer cells trigger angiogenesis by activating genes coding for angiogenesis stimulators and inhibiting genes coding for angiogenesis inhibitors
In large tumors, the middle is usually necrotic due to lack of blood supply
Tissue invasion and metastasis
Cancer cells lose adhesiveness with neighbors, invade nearby tissues, and eventually metastasize around the body via the circulatory system
Tissue invasion: invades local tissue but hasn’t moved out
Metastasis: cancer has moved out of tissue of origin
Deregulating cellular energetics
Tumor cells are shown to reprogram cellular metabolism in order to support neoplastic proliferation
More metabolically active
PET Scan detects unexpected loss of weight
Genome instability and mutation
Increased mutability endows cancer cells with alterations that drive tumor progression
The more the tumor divides, the more mutations occur
Check points are bypassed
Knowing which oncogenes the tumor needs, which have no effect, which harm it, etc. help determine best course of treatment
Avoiding immune destruction
Evasion of the immune system and destruction of cancer cells
Immunotherapy
Tumor-promoting inflammation
It has been shown that inflammatory responses can be tumor-promoting environments
Regulatory T cells down regulate T cells and can inhibit immunotherapy
Obese → ↑ inflammation → ↑ risk of cancer
Colony formation assay
cancer cells lose contact inhibition and will grow on top of one another
Normal cells grow in a monolayer
Often due to the down regulation of integrins so they don’t have to fully flatten and attach before division (cells will show as clumps in tissue culture)
Don’t attach as well to flask/plate
Soft Agar Assay
Bottom layer is harder agar which prevents the cells from attaching to the plate, tumor forms in soft layer, non cancerous cells are suspended and die
Prevents normal cells from growing by inhibiting contact
Feeder layer above cell-containing soft layer
Xenografts
Human cells are put into immunocompromised mice
Hairless mice make it easier for researcher can see the growth of the tumor
Generally injected into the back flank so the mouse can’t chew on the area (if something happens where the tumor breaks through it must be euthanized)
Characteristics of T-ALL mouse model
Splenomegaly, enlarged thyroid, lymph nodes, and liver, due to the undifferentiated T cells being stuck in the organs
in the periphery CD4 and CD8 double positive cells will be out in the periphery
Know how Notch induces cancer formation (molecular pathways)
NotcIC induces T-ALL in mice when expressed in pre-T cells or bone marrow
Mutations found in HD region (embeds notch in membrane) an PEST domain (involved in stability of proteins)
When not working properly the increased expression of MYC will not induce p53, however the main mutation is with notch because in MYC driven tumors there is still 20% relapse
When notch is working properly…
an increased expression will block p53 expression, but when there is an increase in notch, MYC will increase in expression and be able to induce p53
When Notch is not working properly…
the increased expression of MYC will not induce p53, however the main mutation is with notch because in MYC driven tumors there is still 20% relapse
How to titer a virus
Day before plate 6 wells with 5X105 cells per plate, allow to incubate overnight
Remove media from plate and perform the following serial dilution, these numbers can be changed but need a 1:10 dilution, when aliquoted 1.2 ml is frozen down
* Well 1: 1.2 ml viral media
* Well 2: 0.2 ml viral media from above and 1.8 ml normal media
* Well 3: 0.2 ml viral media from above and 1.8 ml normal media
* Well 4: 0.2 ml viral media from above and 1.8 ml normal media
* Well 5: 0.2 ml viral media from above and 1.8 ml normal media
* Well 6: 1 ml normal media
Only need to titer one per viral prep because they should all be the same
After 4 hrs add 2 ml of normal media and collect cells after 48 hours to evaluate
Viral Titering Calculations
when looking at infection curve want to choose a value in the middle, ensures that the number are starting to plateau, after you get numbers do the following calculations
(ml/%) x [X(ml solving for)/% you want to infect] → solve for X to know how much virus to use
Also do not want 100% efficiency because that would not even be possible for bone marrow stem cells, can change based on the cells that you will be infecting
