Bioimaging in 96 well plates

Materials needed:

• Relevant primary and secondary antibodies
• Hoechst 33342 (Molecular Probes catalog no. H-3570)
• Black clear bottom 96 well imaging plates; sterile /w lid (catalog no. 353219)
• 10X Phosphate Buffered Saline (PBS): Prepare a 1X PBS solution by diluting the 10X PBS stock with DI water
• 37% Formaldehyde solution (Sigma-Aldrich catalog no. F-1268 or F-1635): Prepare a 3.7% formaldehyde solution by diluting the 37% formaldehyde stock with 1X PBS that has been freshly diluted from 10X PBS and warmed to 37°C.
• 1% Triton X-100 made up in 10X PBS (prepared and kept at 4°C): Prepare a 0.1% Triton X-100 solution by diluting with DI water so that the final PBS concentration is at 1X.
• 90% Methanol diluted with DI water (prepared and kept at -20°C)
• 3% FBS solution made up in 1X PBS (prepared and kept at 4°C)

Procedure

Sample Preparation:

1. Cells should be plated in the sterile, black 96-well imaging plates (cat.no. 353219) one day in advance of the day of antibody staining. It is recommended that the investigator titrate or determine the optimal number of cells to plate, taking into account the growth rate or proliferation kinetics for the overnight culturing of the cells being analyzed. A cell density that is either too high or too low may result in suboptimal staining. Suggested cell numbers to plate include 8,000- 10,000 cells/well for the cell lines HeLa, A549, HUVEC, HE, A431, CHOK1, MCF7 and U2OS.
2. Cells should be cultured in the wells in a total volume between 100 µl to 200 µl. If cells are to be treated with a compound, the treatment can take place within the well with the investigator determining optimal dosage and exposure times.

Fixation

1. After the overnight incubation, the culture media is removed by turning the plate upside down and "flicking" the liquid out with dabbing on paper towels to remove excess residual liquid. Alternatively, vacuum aspiration or the use of a plate washer can be utilized to remove the culture media, but care must be taken to avoid a loss of cells on the plate bottom.
2. Once the culture media has been removed, add 100 µl per well of a 3.7 % formaldehyde solution that has been pre-warmed to 37°C.
3. Incubate the cells in the 3.7 % formaldehyde solution at room temperature for 10 minutes.
4. After the incubation, remove the formaldehyde solution by "flicking" and dabbing out the residual liquid.
5. Perform a wash by adding 100 µl per well of 1X PBS and then immediately removing the liquid solution by "flicking" and dabbing.
6. Proceed with either the 0.1% Triton X-100 or 90% Methanol permeabilization methods listed below. Guidance as to which method should be selected is detailed on the technical data sheet for the BioImaging Certified antibody to be used.

Permeabilization Method 1: 0.1% Triton X-100

1. After removal of the 1X PBS, add 100 µl per well of a 0.1% Triton X-100 solution.
2. Incubate the cells in the 0.1% Triton X-100 solution at room temperature for 5 minutes.
3. After the incubation, remove the 0.1% Triton X-100 solution by "flicking" and dabbing out the residual liquid.
4. Perform a wash by adding 100 µl per well of 1X PBS and then immediately removing the liquid solution by "flicking" and dabbing.
5. Perform a second wash by adding 100 µl per well of 1X PBS and then immediately removing the liquid solution by "flicking" and dabbing.
6. Proceed with the "Blocking" section.

Permeabilization Method 2: 90% Methanol

1. After removal of the 1X PBS, add 100 µl per well of a chilled 90% methanol solution (the 90% methanol is kept at -20°C prior to adding).
2. Incubate the cells in the 90% methanol solution at room temperature for 5 minutes.
3. After the incubation, remove the 90% methanol solution by "flicking" and dabbing out the residual liquid.
4. Wash twice by adding 100 µl per well of 1X PBS and then immediately removing the liquid solution by "flicking" and dabbing.
5. Proceed with the "Blocking" section.

Blocking

1. After removal of the 1X PBS, add 100 µl per well of a 3% FBS solution.
2. Incubate the cells in the 3% FBS solution at room temperature for 30 minutes.
3. After the incubation, remove the 3% FBS solution by "flicking" and dabbing out the residual liquid.
4. Proceed with the Primary Antibody Staining section.

Primary Antibody Staining

1. After removal of the 3% FBS solution, add 50 µl per well of the diluted primary antibody. Because the optimal antibody concentration can vary with different cells, investigators are encouraged to titrate the antibody over a wide range of concentrations. All antibody dilutions should be performed in the blocking solution (the 3% FBS solution).
2. Incubate the cells with the primary antibody solution at room temperature for 1 hour (in the dark, protected from light if the antibody is labeled with a fluorescent dye).
3. After the incubation, remove the primary antibody solution by "flicking" and dabbing out the residual liquid.
4. Wash three times by adding 100 µl per well of 1X PBS and then immediately removing the liquid solution by "flicking" and dabbing.
5. If the primary antibody was a purified antibody, proceed with the Secondary Antibody Staining section.
6. If the primary antibody was conjugated to a fluorescent dye, add 200 µl per well of 1X PBS containing 2 µg/ml Hoechst 33342.
7. Cover the plate and analyze using an imaging instrument.

Secondary Antibody Staining

1. After removal of the 1X PBS, add 50 µl per well of the secondary antibody at its recommended concentration. Because the optimal antibody concentration can vary with different cells, investigators are encouraged to titrate the antibody. In most cases, 0.25 µg of the secondary antibody in a volume of 50 µl will work well for fluorochromes such as Alexa Fluor^® 488, Alexa Fluor^® 555, Alexa Fluor^® 647, FITC, and APC. All antibody dilutions should be performed in the blocking solution (the 3% FBS solution).
2. Incubate the cells with the secondary antibody solution at room temperature for 1 hour in the dark (protect from light).
3. After the incubation, remove the secondary antibody solution by "flicking" and dabbing out the residual liquid.
4. Wash three times by adding 100 µl per well of 1X PBS and then immediately removing the liquid solution by "flicking" and dabbing.
5. After removal of 1X PBS, add 200 µl per well of 1X PBS containing 2 µg/ml Hoechst 33342.
6. Cover the plate and analyze using an imaging instrument.

For a detailed description of Bioimaging-certified antibodies, please visit our catalog.

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