Assessment of Opsonophagocytic Killing Activity against Bacterial Pathogens
Trascrizione
Thaw one tube of bacterial stock. Pellet bacteria at 6,000 times g for two minutes, and resuspend the cell pellet in OBB at the optimal dilution obtained in the previous step. Pipette 10 microliters of the resuspended bacterial dilution per well in a round bottom 96-well cell culture plate. Then, add 20 microliters of an appropriate antibody or a drug treatment to each experimental well in duplicate.
For control wells, use 1X PBS or OBB, depending on the buffer used for the treatment wells. Shake the sample plate at approximately 90 RPM at room temperature for one hour.
To harvest the HL60-differentiated cells that are treated with DMF three days prior, pellet the cells at 500 times g for three minutes. Discard the supernatant and wash the pellet with at least 10 milliliters of 1XPBS. Pellet the washed cells at 500 times g for three minutes. Discard the supernatant and resuspend the cells in OBB.
Add sterile undiluted baby rabbit serum at a one to five final volume. After a one-hour bacterial culture, divide each sample into duplicate wells for two groups. Next, add 50 microliters of the HL60 complement mixture to each experimental set of wells and 50 microliters of OBB to the wells of bacteria only. Shake the 96-well plate at 37 degrees Celsius for one hour.
To plate the samples, dilute each well with OBB at a one to five final volume so that each sample has a volume of at least 50 microliters. Next, pipette 50 microliters of each sample directly onto a designated area of a bacterial culture plate, ensuring adequate spacing between samples. Cover, and allow samples to dry for approximately 15 minutes at room temperature.
Invert the plates and culture overnight at 30 degrees Celsius. Alternatively, culture plates in anaerobic jars, to test whether anoxic conditions affect the bacterial growth or to control for morphology. After overnight culture, count the colonies in each designated sample area and proceed to analyzing data by comparing the number of live cells in each set to the corresponding controls.
The most difficult aspects of establishing this technique for the first time will likely be optimizing the starting CFUs of the bacterial stock, as well as ensuring the HL60 cells are effectively differentiated.
