Immunofluorescent Staining of Live Cells

This protocol describes the procedure for direct immunofluorescent (IF) staining of live cells in culture using BioLite™ Antibodies. All steps should be performed in a sterile working environment using fresh culture medium.

• BioLite Antibody
• Cell Culture Medium
• Pluripotent Stem Cells

• When using a primary antibody for the first time, it is recommended that the antibody be titrated for optimal performance specific to your cell type.
• Cells can be grown, treated, and stained directly in multi-well plates.
• Only open the antibody in a biological safety cabinet to prevent possible contamination.
• Protect fluorescent conjugates from light.

• 1. Refer to the Technical Data Sheet for the recommended starting concentration, and titrate the primary antibody for optimal performance on your cell type.
• 2. Dilute the primary antibody in fresh cell culture medium to the determined optimum concentration.
• 3. Aspirate the culture medium from the well of cells to be stained, and add the diluted antibody directly to the well of live cells.
• 4. Incubate the cells in a 5% CO₂ incubator at 37°C for 30 minutes.
• 5. Aspirate the primary antibody and gently wash the cells twice with fresh culture medium.
• 6. Add fresh cell culture medium to the well.
• 7. Examine the cells using a fluorescence microscope and appropriate filters.
• 8. Return cells to the incubator and continue to culture as normal.