What is a hemocytometer and explain how it works?
An instrument used in the manul counting of cells.
Through capillary action, a sample of cells from the cell suspension are drawn into the chamber betweenthe cover slip and the hemocytometer. Using the calibrated grid (9 larger squares: 3x3, which are further divided into 25 squares: 5x5).
**1mm x 1mm x 0.1mm = 0.1mm = 1x 10^-4mL = 0.1uL
Once the cells are counted, the total number of cells per mL can be determined.
Explain how a Counter counter works
The counter is placed into a sampling orifice. The submerged portion of the counter has an opening flanked by 2 electrodes that create an electrical field. The un-submerged end is connected to a vacuum. When the vacuum is turned on, particles that pass by the opening will disrupt the electrical field. Each time the field is disrupted, it is counted by the Coulter counter.
The apparatus is able to set upper and lower bound limits on the particle size. Can be very specific to cell size.
Name and explain two indirect methods of cell counting.
Protein determination - this measures the total amount of protein in the cell culture. First you must lyse the cells, add in the NaOH and coomassie blue. This produces a blue solution which can be observed using a spectrophotometer and compared against a standard
Glucose determination - Measure the changes in the glucose levels in the medium with respect to cell density (glucose is commonly used in culture medium)
**can also measure lactate production or oxygen consumption (these are factors that also indicate glucose utilization)
This technique is good for immobilized cells in bioreactors.
What are methods of determining cell viability?
Cell Membrane Integrity
Explain how colony formation works
This is a test of cell viability by directly measuring a cell's ability to grow.
The cells are treated with trypsin and versine and seeded onto petri dishes with a control and different test variables.
Explain how the cell membrane integrity test works
There are 2 types: dye exclusion and dye retention
Dye Exclusion: this assay tests the ability of the cell membrane to exclude bulky charged molecules
The most common dye used is trypan blue and the dye only penetrates the membrane of dead cells. The number of stained cells relative to non-stained indicates the viability of cell culture
Dye Retention: There are 2 markers - lactate dehydrogenase (LDH) and carboxyfluorescein diacetate (CFDA)
LDH marker - the enzyme is generally intracellular and if there is a damage to the cell membrane. Cells that are not viable (with damage to membrane) will leak LDH into the medium. The assay checks for LDH activity within the medium.
CFDA marker - CFDA is a non-polar, non-fluorescent dye which diffuses into cells rapidly (due to non-polarity). Within the cell, itis converted to CF, which requires an intact intracellular environment. CF is polar and fluroscent and because of this diffuses out of the cell very slowly. The measure of fluorescence checks for the viable cells.
Explain how metabolic activity works in cell viability assay
There are 2 tests: Tetrazolium and Resazurin reduction assay
Tetrazolium Assay - is a measure of oxidative metabolism (mitochondrial activity). The dye MTT is reduced by succinate dehydrogenase which turns the yellow MTT into a blue coloured product.
Some cells do not have enough succinate dehydrogenase for MTT assay to be effective
Resazurin Reduction Assay - A non-fluorescent dye that permeates the cell membrane and is reduced by mitochondrial enzymes Diaphorases. This turns a dark blue colour into a purple/pink product which is detected using a spectrophotometre.