STAINING PROCEDURE FOR CORRELATION OF SURFACE ANTIGEN EXPRESSION SIMULTANEOUSLY WITH DNA CONTENT

MATERIALS:

 

1. 2% formaldehyde solution (preparation method attached)

2. 1 X PBS (without sodium azide and serum)

3. 1 X PBS + 2% newborn calf serum + sodium azide (buffer)

4. Polyoxyethylensorbitan monolaureate (Tween 20)

5. Human AB serum (HAB, heat-inactivated)

6. Propidium iodide (PI)(e.g., from Calbiochem)

7. Ribonuclease A (RNase)(e.g., from Sigma)

8. 7-amino-Actinomycin-D (7-AAD, e.g., from Calbiochem)

6. 37C water bath, refrigerated centrifuge.

METHOD:

Staining of surface antigens

1 x 106 PBS-washed cells from a single cell suspension are pelleted in a 12 X 75 mm culture tube. Then the staining proceeds as described in A1-3 for FITC-labelled antibodies and in B1-3 for simultaneous staining with FITC and PE-labelled antibodies.

Fixation

The pellet is resuspended in 0.875 ml of cold PBS and the suspension is mixed gently. Then, 0.125 ml of cold 2% formaldehyde solution is added and the mixture is immediately vortexed briefly. The suspension is incubated for at least 30 min or for up to 1 h at 4C, centrifuged for 5 min at 250g, then the supernatant is removed.

Permeabilization

The pellet is gently resuspended in room temperature Tween 20 solution (0.2% in PBS) and the mixture is incubated for 15 min in a 37C water bath. One ml of buffer is added and the suspension is spun for 5 min at 250g, then the supernatant is removed.

Staining of DNA

Proceeds as described in A5 for samples that are stained with FITC-labelled antibodies on the surface and in B5 for samples with dual-color surface labelling.

 

A. Procedure for staining of surface antigens with FITC-conjugated antibodies and DNA staining with PI

1. Add 50 microliters of HAB to your cell pellet, mix and wait for approximately 1 min.

2. Add 50 microliters of buffer and the appropriate amount of antibody, vortex briefly and incubate for 30 min at 4C in the dark.

3. Wash twice with 1 ml of buffer by centrifugation at 250g for 5 min.

4. Fix and permeabilize as described under METHOD.

5. Resuspend samples in 1 ml of buffer containing 10 micrograms/ml of PI and 11.25 Kunitz units of RNase and incubate for at least 30 min at 4C in the dark. Keep them in the staining solution until analysis on the flow cytometer.

B. Procedure for staining of surface antigens with FITC and PE-conjugated antibodies and DNA staining with 7-AAD

1. Add 50 microliters of HAB to your cell pellet, mix and wait for approximately 1 min.

2. Add 50 microliters of buffer and the appropriate amount of FITC and/or PE-conjugated antibody, vortex briefly and incubate for 30 min at 4C in the dark.

3. Wash twice with 1 ml of buffer by centrifugation at 250g for 5 min.

4. Fix and permeabilize as described under METHOD.

5. Resuspend samples in 1 ml of buffer containing 10-25 micrograms/ml of 7-AAD and incubate for at least 20 min at 4C in the dark. Keep them in the staining solution until analysis on the flow cytometer.

Note: it is not essential to use 50% HAB for surface staining, but it can prevent problems with Fc binding of antibodies. However, do not use HAB for staining of immunoglobulin chains and when you are using antibodies that are directed against Fc receptors (e.g. CD16).

7-AAD has to be used for DNA staining in combination with PE-labelled antibodies due to the spectral emission overlap between PE and PI.

PREPARATION OF 2% FORMALDEHYDE STOCK SOLUTION (2 METHODS)

METHOD 1:

Formaldehyde preservative – 2% formaldehyde solution in protein-free phosphate-buffered saline (PBS).

Prepare as follows:

Add 2 g paraformaldehyde powder (e.g., Sigma, St. Louis, MO) to 100 ml of 1 X PBS. Heat to 70C (do not exceed this temperature) in a fume hood until the paraformaldehyde goes into solution (note that this happens quickly as soon as the suspension reaches 70C). Allow the solution to cool to room temperature. Adjust to pH 7.4 using 0.1 M NaOH or 0.1 M HCl, if needed. Filter and store at 4C protected from light.

METHOD 2:

Formaldehyde preservative – 2% formaldehyde solution in protein-free PBS.

Prepare as follows:

10% formaldehyde* 20 ml
10 x PBS 10 ml
Distilled water 70 ml

* 10% formaldehyde solution (e.g., Polysciences, Warrington, PA, ultrapure, Cat.#04018), depolymerized paraformaldehyde, EM grade, methanol-free solution.