Cell proliferation BrdU (JUMC)

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SOP for Cell Proliferation, BrdU (JUMC)


Reagents:
• Cell Proliferation ELISA, BrdU Kit ROCHE, Cat. No. 1 647 229

Kit contents :
 BrdU labeling reagent [10mM 5-bromo-2’-deoxyuridine in PBS, pH 7.4];
 FixDenat 2 × 100ml (Ready-to-use);
 Anti-BrdU-POD (Lyophilizate, stabilized Monoclonal antibody from mouse-mouse hybrid cells, clone BMG 6H8, Fab fragments) conjugated with peroxidase (POD);
 Antibody dilution solution 100 ml (Ready-to-use);
 Washing buffer 100ml PBS 10×conc.;
 Substrate solution 100ml TMB (tetramethyl-benzidine) Ready-to-use.

• Culture medium
• Didistilled water

Preparation of working solutions:
.
• BrdU labeling solution
(bottle 1) Dilute BrdU labeling reagent 1:100 with sterile culture medium (resulting concentration:100 µM BrdU).
For one 96-well MP containing 100 µl/well culture medium 1 ml BrdU labeling solution is required. If the cells have to be cultured in 200µl/well, add 20µl/well BrdU labeling solution.

The undiluted BrdU labeling Reagent (1000 ×):
At 2-8°C for several months protected from light.
(bottle2)The diluted BrdU labeling reagent:
At 2-8°C stable for several weeks store protected from Light.
For long-term storage it is recommended to store the BrdU labeling solution in aliquots at -15 to -25°C.

Labeling of cells.

• Anti-BrdUPOD stock solution
(bottle 3) Dissolve Anti-BrdUPOD in 1.1 ml double dist. water for 10 min and mix thoroughly.
At 2-8°C for several months. For long-term storage it is recommended to store the solution in aliquots at -15 to -25°C.
Stock for the preparation of the Anti-BrdU POD working solution.

• Anti-BrdUPOD working solution
(bottle 4) Dilute Anti-BrdU-POD stock solution 1:100 with antibody dilution solution.
For one 96-well MP dilute 100µl Anti-BrdU-POD stock solution in 10 ml antibody dilution solution (bottle 4).
Prepare shortly before use ! Do not store!
Binding of the POD labeled anti-BrdU antibody.

• Washing solution
(bottle 5) Dilute Washing buffer concentrate 1:10 with double dist. water.
For one 96-well micropalte (MP) dilute 10 ml Washing buffer concentrate (bottle 5) with 90 ml double dist. water.
At 2-8°C for several weeks For the removal of unbound antibodies.

Procedure:

1. To culture cells 70% of confluency together with various dilutions of test substance in a 96-well MP in a final volume of 100 µl/well in a humidified atmosphere at 37°C.

2. Add 10 µl/well BrdU labeling solution if the cells were cultured in 100µl/well (final
concentration:10µM BrdU) and reincubate the cells for additional 3 to 24 h at 37°C (if the cells were cultured in 200µl/well, add 20 µl/well BrdU labeling solution).

3. Removal of labeling medium:
-For adherent cells
• Remove labeling medium by tapping off or suction.
-For suspension cells:
• Centrifuge the MP at 300xg for 10 min, room temperature and remove the labeling medium by flicking off or suction using a canulla.
• Dry cells using a hair-dryer for about 15 min or, alternatively, at 60°C for 1 h.

4. After removal of the labeling medium and drying of the labeled cells the dry cells can be stored for up to one week at 2–8°C.

5. Add 200 µl/well FixDenat (bottle 2) to the cells. Incubate for 30 min at 15–25°C.

6. Remove FixDenat solution thoroughly by flicking off and tapping.
7. Add 100µl/ well anti-BrdU-POD working solution. Incubate for approx. 90 min at 15–25°C.

8. Remove antibody conjugate by flicking off and rinse wells three times with 200µl–300 µl/well Washing solution (solution 4).

9. Remove Washing solution by tapping.
Add 100 µl/well Substrate solution. Incubate at 15–25°C until color development is sufficient for photometric detection (5-30 min).

10. Without stop solution:
• Measure the absorbance at 370 nm (reference wavelength: approx. 492 nm).
With stop solution:
• Add 25 µl 1M H2SO4 to each well and incubate the MP for approx. 1 min on the shaker at 300 rpm (or mix thoroughly). Measure the absorbance at 450 nm (reference wavelength: 690 nm).

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