This study investigates the role of TNFα in inducing apoptosis in serum-deprived mouse fibroblasts and the protective effects of TNFα-specific antibodies. The methodology includes measuring luminescence intensity to assess the effectiveness of the antibodies in preventing apoptosis.
Take a multi-well plate containing mouse fibroblasts in serum-deprived media. Add serial dilutions of TNFα-specific antibodies.
Add TNFα — an inflammatory cytokine.
Incubate. Serum deprivation induces cellular stress, making the cells susceptible to TNFα-mediated apoptosis.
TNFα binding to its receptors on the serum-deprived cells triggers intracellular signaling cascades, activating the caspase cascade and inducing apoptosis.
However, antibody binding to TNFα prevents its receptor binding, inhibiting caspase activation.
Post-incubation, add a reagent mixture containing detergent, a proluminescent caspase substrate, and a luciferase enzyme along with ATP and magnesium ions. Mix and incubate.
The detergent permeabilizes the membranes, releasing caspases from TNFα-induced cells.
The bioluminescent substrate contains a caspase recognition site. Specific caspases cleave the substrate, generating aminoluciferin.
Luciferase oxidizes its substrate, aminoluciferin, resulting in light emission.
Measure luminescence intensity to detect a decline in luminescence with increasing antibody concentrations, indicating the potency of the TNFα specific-antibodies to neutralize TNFα and prevent TNFα-induced apoptosis.
Add 50 microliters of cell suspension to the internal 60 wells of the assay plate, starting from the column 2 to the 11th. Mix cell suspension by pipetting to add aliquots to the microplate. Immediately, transfer 50 microliters of the mapped dilutions to the assay plate.
For parallel lanes, dispense dilutions by turning around the micropipette. The mapped dilutions can be dispensed into the wells in the following order. Controls are dispensed in the surrounding wells.
Dilute TNF-alpha with 500 microliters of ultrapure water. Mix by vortex mixer at 2,200 RPMs. Transfer to a 15 mL tube, and finally, dilute to 40 nanograms per mL with assay culture medium. Dispense 50 microliters of the apoptosis induction solution into the 60 internal outside wells. Dispense microliters of the apoptosis induction solution into the 60 internal assay wells. Finally, incubate for 16 hours.
On fifth day, reconstitute substrate with its buffer solution as directed in manufacturer's instructions. Mix by inverting five times. Dispense substrate solution, and add 50 microliters of the substrate solution to each sample well in the assay plate. Also, add substrate to controls. Agitate the assay in a vortex mixer at 400 RPMs for three minutes.
Take out the assay plate from the mixer and incubate at room temperature. Turn on a plate-reader spectrophotometer. Open the Softmax Pro software or any equivalent program. Load microplate into the spectrophotometer and adjust the following parameters.
Activate the luminescence function on the rig type into endpoint function. Adjust the rig area excluding columns 1 and 12. Select the plate type to a 96-well standard clear bottom microplate. Alter the integration time up to 1250 milliseconds. Adjust the shake time for the microplate to 10 seconds. Select the wall in which reference substance, analytical substance, and control sample will be placed. Begin to read.
繁體中文
