Cell Culture
Are our cells adherent or suspended? Why?
Adherent - they "sit down" on the bottom of our tissue flasks
Name three cell lines that our lab has
What is the physical restraint of a mouse called?
Scruffing
What is forward scatter and side scatter on a flow cytometer? What do they measure?
Forward scatter: how large a cell is
Side scatter: how complex or granular a cell is
What are phagocytes and what do they do? Give an example of a cell type
Phagocytes "eat" foreign bodies and cell debris to clear these from the body. Two examples are neutrophils and macrophages.
What is trypsin and what does it do?
Trypsin is a digestive enzyme that helps to break down the cell's ability to adhere to the flask.
Name our three triple negative breast cancer cell lines
D2F2, 4T1, E0771
Name three out of the five primary types of injections you can perform in a mouse
Subcutaneous, intramuscular, retro-orbital, tail vein, intraperitoneal
How can you determine live from dead cells? How does the stain work?
Using AOPI stain or a viability stain on the flow cytometer. On the flow cytometer, the stain binds to the dead cells so the live cells do not fluoresce.
What cell types primarily are involved with allergic reactions?
Eosinophils or mast cells
What percent FBS is in our SDME media? What does FBS stand for?
10%; fetal bovine serum
What is one way that you can introduce DNA using lipids into a cell to be expressed?
Transfection
What are our main strains of mouse?
FVB, B6, BALB/c
How can you look at multiple targets using the flow cytometer? What allows us to do this?
The cytometer has multiple lasers that correspond to different excitation/emissions wavelengths that are unique to individual fluorophores. Each target antibody can be conjugated to different fluorophores for visualization.
How might you measure an organisms response to a vaccine?
Measure B cell response via antibody production.
Measure T cell response via activation when exposed to the antigen which was vaccinated against (such as an assay like the Elispot)
Thaw fast, freeze slow (in a freezer buddy!)
What are primary cells? Name a primary cell type that we work with
Primary cells are cells taken directly from tissue to be cultured. One example of this is bone marrow stem cells to derive macrophages.
Name 5 out of the 8 strains included in the diversity outbred background
B6, PWK, WSB, A/J, NZO, NOD, CAST, 129
Glucocorticoid Induced Leucine Zipper. Higher expression of this protein has been associated with better overall survival in triple negative breast cancer patients.
Name two myeloid lineage immune cells. How do they differ?
Macrophages: have many roles, including phagocytosis and antigen presentation. More common in the body
Dendritic cells: primarily strong antigen presenters, very few in the body
What is the recipe for our freezing media?
70% media
20% FBS
10% DMSO
How can you make sure that your cells keep a plasmid after being introduced?
Use a plasmid with antibiotic resistance and plate the cells with those antibiotics.
Name two approved methods of euthanasia.
Cervical dislocation, bilateral pneumothorax, exsanguination, barbituate overdose, CO2 inhalation, essential organ removal.
What is LILRB4? Why are we interested in this checkpoint molecule and where is it expressed?
Leukocyte immunoglobulin-like receptor B4. This checkpoint molecule is expressed on myeloid cells and is hypothesized to decrease T cell activation through downregulation of MHCs and CD80/CD86 (costimulatory molecules)
What does MHC stand for? What are the roles of MHC I vs MHC II?
MHC: major histocompatibility complex
MHC I: presents intracellular antigen
MHC II: presents extracellular antigen