Flow Cytometry Analysis:
Prepare the following solutions before proceeding:
Phosphate buffered saline (PBS)
2N HCl, 0.5% Triton X-100
PBS containing 0.05% Tween-20
PBS containing 1% BSA (PBS/BSA)
10mg/ml Propidium iodide (PI)
As a positive control, BrdU labeled cells maybe obtained from Phoenix Flow Systems (http://www.phnxflow.com), catalogue number ACNC12.
1. Add BrdU to the cell suspension in culture medium to a final concentration of 10 umol/L and incubate for 30 minutes in a CO2 incubator at 37C.
2. Wash cells twice with PBS/BSA, and resuspend in PBS
3. Add cells slowly into 5ml of 70% ethanol at -20C, mixing continuously (vortex preferred). Incubate on ice for 30 minutes.
4. Centrifuge at 500g for 10 minutes, decant supernatant, and resuspend cell pellet.
5. Add 2ml 2N HCl, 0.5% Triton X-100 and incubate the cells for 30 minutes at room temperature (preferably on a rocking platform).
6. Centrifuge at 500g for 10 minutes, decant supernatant and resuspend in 3 ml 0.1M Na2B4O7, pH 8.5
7. Centrifuge at 500g for 10 minutes, decant supernatant and resuspend the cells in PBS/BSA + 0.05% Tween 20. Adjust cell concentration to 1 x 10(7)/ml
8. Aliquot 100ul of cell suspension into required number of 12 x 75mm tubes.
9. Incubate the cells with the anti-BrdU antibody at the recommended dilution for 30 minutes at room temperature.
10. Add 2 mls PBS/BSA and centrifuge the cells at 1000rpm for 5 minutes.
11. If a secondary antibody layer is required then decant the wash and incubate the cells with the secondary antibody for 30 minutes at room temperature. If no secondary antibody layer is required then proceed to step 13.
12. Wash the cells after the secondary antibody layer by repeating step 10.
13. Decant off the wash and add 1ml PBS containing 10ug/ml PI (Dilute the 10mg/ml solution of PI 1/1000 in a suitable volume of PBS)
14. Analyse cells by flow cytometry following the manufacturer's instructions. The PI should be read on the appropriate channel set to the Peak/Area and not log scale.
IMMUNOHISTOLOGY
Formalin-fixed paraffin-embedded tissue sections:
Clone BU1/75 (ICR1) can be used for labelling paraffin-embedded tissue sections fixed in formalin. Pretreatment of tissues with heat-induced epitope retrieval using 10mM citrate buffer pH6.0 is recommended. Pretreatment of tissues with proteinase K should be avoided.
