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Millipore Technical Publications

Protocol: Immunocytochemistry Adherent Cells MAB3299 Protocol for Adherent Cell Cultures

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Sample Preparation

To grow, treat and analyze cells in microplate or 24 well coverslip:

1. Seed 5,000 to 10,000 cells per well and treat with apoptosis inducing agents after 24 to 48 hours.
2. Remove medium, add 200-500 microliters of fixative (80% methanol in PBS made fresh) and incubate at room temperature for 30 minutes.
   

For non-fixed cells grown outside microplate:

1. Transfer 1,000 to 10,000 non-fixed cells to each well or prepared cytospin.
2. Centrifuge plate at 200g for 5 minutes. Remove medium, add 200 microliters of fixative (80% methanol in PBS) and incubate at room temperature for 30 minutes. Transfer to cytospins or wells when ready to begin assay.

Assay Instructions

1. Following transfer and fixation of cells as noted above, remove fixative.
2. Dry plates by floating in waterbath at 37ºC for 20 minutes, or keep at room temperature for 1-2 hours. This will allow attachment of cells to the plate. Note: Once plates are dried they can be store, dry, at room temperature overnight if desired.
3. Add 50 microliters of Formamide to each well and incubate at room temperature for 10 minutes.
4. Heat plates to 75ºC in a circulating waterbath for 10 minutes or in an oven (without lid) for 20 minutes. This will denature DNA in apoptotic cells.
5. Cool plates in refrigerator for 5 minutes, then remove formamide.
6. Add 200 microliters of 3% nonfat dry milk in distilled water (w/v).
7. Incubate plates at 37ºC for 1 hour to block non-specific binding sites.
8. Remove milk and add 100 microliters of Antibody Mixture (1:10-1:20 dilution, diluted in 3% non-fat milk) to each well.
9. Incubate plates at room temperature for 30 minutes.
10. Wash plates 3 times with 1X Wash Buffer (PBS-T: PBS plus 0.05 tween-20), using 250-1000 microliters of wash buffer per well each wash.
11. Add diluted secondary antibody (diluted in 3% non-fat milk), either monoclonal or polyclonal. Dilutions vary 1:100-1:1000 depending on HRP activity; typically 1:100 for monoclonal secondary, 1:300-1:500 for polyclonal are good starting points.
12. Wash 3X with 1X Wash Buffer as in step 10.
13. Add 100 microliters of substrate solution and incubate for 15-60 minutes.
14. Stop the reaction by adding 100µL of stop solution to each well, and read absorbance in standard microplate reader at 405 nm.

References
Frankfurt, O.S. and A. Krishan (2001). Identification of apoptotic cells by formamide-induced DNA denaturation in condensed chromatin. J. Histochem. Cytochem. 49: 369-378.
Frankfurt, O.S. and A. Krishan (2001). Enzyme-linked-immunosorbent assay (ELISA) for the specific detection of apoptotic cells and its application to rapid drug screening. J. Immunol. Methods 153: 133-143.