Determining intranuclear Expression by Flow Cytometry

Description

To determine the Oct4 protein expression of ES cells as a measure of their differentiation

status.

Reagents

· Mouse anti-mouse Oct3/4 antibody (IgG1 isotype) (Transduction Laborotories)

· Goat anti-mouse IgG (Fc specific) FITC Conjugate (Sigma, F-2772)

· Hank’s Buffered Saline Solution with 2% FCS (HF)

· Coulter Permeabilization Kit (Beckman Coulter)

Method

1. Trypsinize cells (10⁶) and generate a single cell suspension in 100 mL of HF

2. Fix with reagent 1 (100 mL) of the Coulter Permeabilization kit for 15 min at RT.

3. Wash with 1 ml of HF

4. Permeabilize with 100 mL of reagent 2 from the kit for 5 min at RT.

5. Add 1 mL of the Oct4 Ab and incubate at RT for 20 min.

6. Wash twice with 1 ml of HF

7. Add 1 mL of anti-mouse FITC conjugated IgG secondary antibody and incubate for 20 min at RT.

8. Wash twice with 1 ml of HF

Re-suspend in 0.5-1 mL of HF for final analysis on flow cytometer