Cell Culture
Are our cells adherent or suspended? Why?
We have both! Cell culture cells are adherent - they "sit down" on the bottom of our tissue flasks.
Bone marrow starts out as suspension but sits down.
Lymphocytes are in suspension.
Name 2 lab chores that need to be completed every week.
Liquid nitrogen, flush eyewash station
What is the initial physical restraint of a mouse called?
Scruffing
What are two (2) ways to measure protein levels
1) Western blot
2) Flow cytometry
How do we measure immune response in mice?
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).
Tumor shrinkage
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.
Does the lab recycle?
YES!
Do we have transgenic mice? If so, what transgenes?
YES!
Neu(+/-), d16HER2(+/-), TSC2(+/-), LILRB4(+/-)(-/-), NeuTxLILRB4(+/-)(-/-)How can you determine live from dead cells? Do we gate live or dead cells?
Using AOPI stain or a viability stain on the flow cytometer. On the flow cytometer, the stain binds to the dead cells so only the dead cells fluoresce.
They were isolated from the 'Thymus' and the 'Bone marrow'; hence 'T' and 'B' cells.
Lymph nodes, spleen, peripheral blood, thymus, bone marrow (immature).
What percent FBS is in our SDME media? What does FBS stand for?
10%; fetal bovine serum
What are the lab chores that should be completed on a monthly basis?
Change the water baths, change water in incubators
Name 2 methods of approved euthanasia.
CO2 inhalation, cervical dislocation, exsanguination, bilateral pneumothorax, barbiturate overdose, vital organ removal.
What is GILZ and why do we care?
Glucocorticoid Induced Leucine Zipper.
Because it regulates inflammation & hypoxia in response to cortisol/GCs, which leads to favorable outcomes in cancer.
What are phagocytes? What do they do?
Extra credit: name 2 examples.
Phagocytes "eat" foreign bodies and cell debris to clear these from the body. Two examples are neutrophils and macrophages.
Thaw fast, freeze slow (in a freezer buddy!)
What does the lab recycle? And where?
1) paper - outside or NEW lab recycle bin
2) labeled plastics - outside or NEW lab recycle bin
3) metal (including foil) - outside or NEW lab recycle bin
4) plastic wrap - Dr. Jacob's office (to Meijer)
Name 5 out of the 8 strains included in the diversity outbred background
129, A/J, B6, CAST, NOD, NZO, PWK, WSB
What is LILRB4 and where is it expressed?
Leukocyte immunoglobulin-like receptor B4. A checkpoint molecule expressed on myeloid cells.
Extra credit: It is hypothesized to decrease T cell activation through downregulation of MHCs and CD80/CD86 (costimulatory molecules)
Name 2 myeloid lineage 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 BMDM? What do they require to differentiate? What is their "panic button"?
Bone Marrow Derived Macrophages. Macrophage-colony stimulating factor (M-CSF); LPS
What chore happens once/semester? Why is it a chore?
Defrosting freezers.
We need to schedule time in backup freezers downstairs.
Name 2 of the 5 methods of injection in a mouse.
Subcutaneous, intravenous (tail), intraperitoneal, intramuscular, retro-orbital.
What is TSC and why do we care?
Tuberous sclerosis complex; it is a tumor suppressor gene up-stream of mTOR. Mutations are linked to various pediatric cancers, including in the kidneys and brain.
What does MHC stand for? What are the roles of MHC I and MHC II?
MHC: major histocompatibility complex
MHC I: presents intracellular antigen
MHC II: presents extracellular antigen