Lab Stuff Flashcards
How do you use a haemocytometer?
- Prepare cell suspension, and mix with trypan blue to stain
- Load into heamocytometer chamber, allowing capillary action to draw suspension under coverslip
- Count cells under microscope
How do you count cells under a microscope for haemocytometry?
Live cells do not take up Trypan blue, whereas dead cells do.
Only count unstained cells
How do you calculate viable cell count (formula)?
(Number of live cells x Dilution factor) / Volume of square
How do you calculate the volume of cells required to achieve correct cell density?
Same as C1V1 = C2V1 but with density
How does MTT proliferation assay work?
Measures cell proliferation by assessing metabolic activity
How does MTT assay assess metabolic activity?
Mitochondrial reductase enzymes in live cells convert the yellow MTT to purple formazan
What are Th1 and Th2?
Subsets of T helper cells
What cytokine does Th1 produce?
IFN-y
What cytokines do Th2 produce?
IL-4, IL-5, IL-13
What is the immunity type associated which each T helper cell?
Th1 - cell-mediated immunity (protection against intracellular viral/bacterial infection)
Th2 - humoral immunity (stimulates B-cell antibody production, often associated with allergens/parasites)
What can ELISA be used to do?
ELISA can measure the cytokines in a sample
How can ELISA measure cytokine levels?
The main format used is sandwich ELISA, in which the cytokine is captured between two antibodies.
Enzyme-linked antibody binds cytokine → Colour change via enzymatic reaction.
What would high IFN-y levels indicate?
A Th1 response, which would be expected for viral or intracellular bacterial infections (cell-mediated immunity)
What would high IL-4/IL-5/IL-13 levels indicate?
A Th2 response, which would be expected for helminth infections or allergic diseases (antibody-mediated immunity)
What do vaccines aim to have?
A balanced Th1/Th2 response, to provide broader protection
For HBV, what are two lab methods for detection?
qPCR and ELISA
What does qPCR detect in HBV?
HBV DNA (viral genome)
What does ELISA detect in HBV?
HBsAg (viral surface antigen)
What is the difference in sensitivity for qPCR vs ELISA?
qPCR has a higher sensitivity, and can detect viral low viral loads.
ELISA requires higher antigen levels for detection
What is the difference in specificity for qPCR vs ELISA?
Both have high specificity, but qPCR is higher- ELISA can give false positives
What is the difference in window period detection for qPCR vs ELISA?
qPCR has an earlier detection window; can detect HBV before serological markers.
ELISA can only detect once surface antigen is present and in high levels
Which out of qPCR or ELISA is better for monitoring?
qPCR, as it can measure and track viral load over time, whereas ELISA mainly just measure the presence of the HPV antigen
What is the difference in cost for qPCR vs ELISA?
qPCR is more expensive than ELISA
What is the clinical use for qPCR for HPV?
Diagnosing acute and chronic infections; monitoring antiviral therapy
