Detection of Intracellular Antigens by Flow Cytometry

实验概要

Fix and Perm  reagents are designed for use with all commercially available flow  cytometers. Alignment and compensation should be performed according to  the manufacturer's instructions. Typical staining and scatter patterns  are shown below in Figure 1.

Clinical Research Applications: Flow cytometric analysis with  monoclonal antibodies has historically been restricted primarily to cell  surface molecules. For this reason, intracellular antigens such as  cytoplasmic or nuclear enzymes, oncoproteins, cytokines,  immunoglobulins, etc., were largely excluded from such analysis. Also  excluded have been cytometric studies designed to address the  cytoplasmic localization of some well established membrane-associated  molecules such as CD3 and CD22. Fix and Perm reagents allow  intracellular antigen analysis with the equivalent ease as cell surface  antigens. The only prerequisite is the availability of suitable antibody  conjugates. Most commercially available monoclonal antibody conjugates  can be used with the FIX & PERM reagents. However, some determinants  are sensitive to the fixation step. This and optimal fixation time may  have to be empirically determined for each antibody conjugate.

主要试剂

Fix & Perm Cell Permeabilization Reagents

Materials Required but Not Included:
Phosphate Buffered Saline containing 0.1% NaN₃ and 5% FBS
12 x 75 mm tubes
Centrifuge
Vortex
Sheath Fluid
Pre-cooled Absolute Methanol

Use of Fix & Perm Reagents:
 Fix and Perm reagents are intended for the fixation (Reagent A) and  permeabilization (Reagent B) of cells in suspension. This procedure  facilitates antibody access to intracellular structures and leaves the  morphological scatter characteristics of the cells intact. Specific  formulations reduce background staining and allow simultaneous addition  of permeabilization medium and fluorochrome-labeled antibodies.

Storage and Stability:
 Fix & Perm reagents should be stored and used at room temperature.  They are stable for the period shown on the package label when stored as  directed. Do not use reagents if a precipitate forms or discoloration  occurs. All antibody combinations should be stored at 2-8°C in the dark.  

Fix & Perm Kit Components should never be frozen.

 Warning: Reagent A of the FIX & PERM Kit contains formaldehyde  which is toxic, allergenic and a suspected carcinogen. Avoid contact  with eyes, skin and clothing. All antibodies contain sodium azide as a  preservative.

实验步骤

1. Permeabilization and Staining Procedure

1)       For each sample to be analyzed add appropriate volume of the conjugated  antibody directed to the cell surface marker(s) of interest and/or the  appropriate isotype control(s) to a 5 ml, 12 x 75 mm tube.

2)      Pipette appropriate volume of adjusted cells (equivalent to 1 x 10⁶ cells) into each tube containing the conjugated antibody or isotype control.

3)      Vortex each tube gently to mix, and incubate for 15 minutes in the dark at room temperature.

4)      Add 100 μl of Reagent A (Fixation Medium) and incubate for 15 minutes at room temperature.

5)      Wash once in 3 ml PBS   0.1% NaN₃   5% FBS.

6)      Centrifuge for 5 minutes at 300-350 x g, aspirate the supernatant, and vortex to fully resuspend the cell pellet.

7)       Add 100 μl of Reagent B (Permeabilization Medium) and the recommended  volume of the FITC- and/or PE- conjugated intracellular antibody or the  corresponding isotype control.

8)      Vortex 1-2 seconds and incubate for 20 minutes.

9)      Wash once in 3 ml PBS   0.1% NaN₃   5% FBS.

10)  Centrifuge for 5 minutes at 300-350 x g and aspirate the supernatant.

11)   Resuspend cells in sheath fluid for immediate analysis or in 0.5 ml of  0.1% paraformaldehyde fixative solution for storage at 2-8ºC in the  dark. Fixed cells should be analyzed within 24 hours.

A modification of this protocol using precooled absolute methanol has  been shown to give better results for certain cell cycle antigens such  as BrdU, Ki-67, and PCNA when using FITC-conjugated antibodies. This  modification is not recommended when using PE-conjugated antibodies.

2. Methanol Modification

1)      For each sample to be analyzed, add 100 μl of adjusted cell volume (equivalent to 1 x 10⁶ cells) to an appropriate 5 ml, 12 x 75 mm tube.

2)      Add 100 μl of Reagent A (Fixation Medium) and incubate for 2–3 minutes at room temperature.

3)      Add 4 ml of pre-cooled absolute methanol (0–4°C) and vortex.

4)      Incubate for an additional 10 minutes at 0–4°C.

5)      Centrifuge for 5 minutes at 300–350 x g and wash with wash medium PBS   0.1% NaN₃   5% FBS.

6)       Add 100 μl of Reagent B (Permeabilization Medium) and appropriate  volume of intracellular antibody(ies) or corresponding isotype  control(s).

7)      Vortex at low speed for 1–2 seconds and incubate for 30 minutes at room temperature.

8)      Wash once in 3 ml wash medium (PBS   0.1% NaN₃   5% FBS).

9)      Centrifuge for 5 minutes at 300–350 x g and aspirate the supernatant.

10)   Resuspend cells in sheath fluid for immediate analysis, or fix in 0.5  ml of 0.1% paraformaldehyde and store at 2–8°C in the dark. Fixed cells  should be analyzed within 18 hours.

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