Evaluating the Intracellular Distribution of Virus-Derived Peptide-Modified Antibody Conjugates

Treat target TF-1a cells with 200 nanomolar 7G3-cholic acid-antibody conjugate. Include cells treated with modified control monoclonal antibodies. Incubate 5 x 10⁶ antigen-positive cells with the conjugates at 37 degrees Celsius for one hour. Then remove the supernatant and use one milliliter of ice-cold PBS to wash the cells three times.

Add fresh medium and incubate the cells at 37 degrees Celsius for an additional hour. Next, add 0.5 milliliters of PBS containing 0.25% trypsin and EDTA, and incubate the cells at 37 degrees Celsius for up to 30 minutes. Then, use 1.5 milliliters of RPMI-1640 medium with 10% FBS to neutralize the trypsin.

Centrifuge the cells at 500 times g for five minutes. Remove the supernatant, and use 0.5 milliliters of ice-cold PBS to wash the cells three times again. Add 0.5 milliliters of 1% PFA and 1% sucrose in PBS to the cells, and place them on ice for 30 minutes to fix them.

Then use 0.5 milliliters of ice-cold PBS to wash the cells three times, and centrifuge the samples at 250 times g for five minutes. Next, add 0.15% Triton X-100 in PBS to the cells, and permeabilize them on ice for five minutes. Then with ice-cold PBS, wash the cells and repeat the centrifugation.

Suspend the cells in 0.1 milliliters of PBS containing two micrograms per milliliter of an anti-murine Fc secondary polyclonal antibody conjugated to AlexaFluor 647, and incubate the samples in the dark at room temperature for one hour. Centrifuge the cells at 250 times g for five minutes, and use 0.5 milliliters of ice-cold PBS to wash the cells three times.

Then resuspend the cells in 0.5 milliliters of PBS. Add 10 micrograms per milliliter of propidium iodide to the cells. Then, use mounting medium to mount 1 x 10⁵ cells onto glass slides before covering the sample with a glass coverslip. Examine the cells with a Plan Apo 60X oil immersion objective, numerical aperture 1.42, on an inverted laser-scanning confocal microscope.

Detect PI fluorescence using the 488-nanometer argon laser and the spectral scanning prism set for 600 to 650 nanometers. For AF 647 fluorescence, use the 633-nanometer helium-neon laser and the spectral scanning prism set for 650 to 700 nanometers. Collect images from P1 and AF 647 sequentially, 1024-by-1024 pixels with 2X line averaging taken at 0.5-micron intervals through the entire cell thickness. Present images as Z projections.

To analyze the cells, evaluate and record whether intracellular fluorescence in the cytoplasm is grouped and near the cell surface or diffuse and homogeneous. Also, evaluate the relative fluorescence intensity per cell.