Staining Intracellular Antigens for Flow Cytometry

2018-08-02Author:adminpraise:0

Introduction

A modification of the basic immunofluorescent staining and flow cytometric analysis can be used for simultaneous analysis of surface molecules and intracellular antigens at the single-cell level by flow cytometry. Typically, cells are fixed with formaldehyde to stabilize the cell membrane, then permeabilized with detergent or ethanol to allow antibodies against intracellular antigens, to access to stain intracellularly.


Protocol-- intracellular (cytoplasmic) proteins

The following protocol is intended for intracellular antigens analysis at the single-cell level. In this protocol, permeabilization after fixation results in pores in the cell membrane. Thus, antibodies enter into cell cytoplasm. Considering surface staining is needed in some experiment, please follow the Staining Cell Surface Targets for Flow Cytometry protocol firstly, then fixate and permeate the cells and stain the intracellular proteins.

 

1. Prepare cells(More detail you can view Sample Preparation for Flow Cytometry)

1) Collect the cells. Collect the cells which are stimulated and interdicted (refer to the literature), add cell staining buffer (or PBS with 0.1% BSA) to make cells re-suspended with the concentration of 1X107 /mL.

2) Aliquot 100 µL cell suspension (about 1X106 cells) and add it into the flow tube.


2. Fixation  

1) If the cell surface staining is needed, follow the  Staining Cell Surface Targets for Flow Cytometry  protocol firstly, and then add 100 uL 1X Fixation /Permeablization buffer to the tube make the cells resuspended, then incubate the cells at room temperature, protect from light for 30 min.

2) Centrifuge at 300 g for 5 min at room temperature. Discard supernatant.


3. Permeabilization

1) Add 2 mL 1X permeabilization buffer to re-suspend the cells, centrifuge at 300 g for 5 min at room temperature. Discard supernatant.

2) Repeat washing one time, centrifuge at 300 g for 5 min at room temperature. Discard supernatant.

3) Add 1 mL 1X permeabilization buffer, then incubate the cells at 4, protecting from light for 30 min. Centrifuge at 300 g for 5 min at room temperature. Discard supernatant.


4. Intracellular staining

1) Add 100 uL 1X permeabilization buffer to re-suspend the cells, and add the Fluorescein-labeled antibody according to datasheet. Mix them and incubate the cells at 4, protecting from light for 30 min.

2) Add 2 mL of 1X permeabilization buffer to re-suspend the cells, centrifuge at 300 g for 5 min at room temperature. Discard supernatant.

3) Add 2 mL of cell staining buffer (or PBS with 0.1% BSA) to re-suspend the cells, centrifuge at 300 g for 5 min at room temperature. Discard supernatant.

4) Add 0.5 mL of cell staining buffer (or PBS with 0.1% BSA) to re-suspend the cells. Detect and analyze by flow cytometry.

 

Notice

1. If the cell surface staining is needed, follow the  Staining Cell Surface Targets for Flow Cytometry  protocol firstly, then fixed and Permeabilizated.

2. It’s suggested that the type control should be used with intracellular antigens staining.