CombinedFlowCytometricMeasurementofTwoCellSurfaceAntigens

INTRODUCTION

Flow cytometry is frequently used to assess nucleic acid content in individual cells. Based on DNA content alone, however, cells in the quiescent G₀ phase cannot be discriminated from cellsin the proliferative G₁ phase, as DNA content remains constant until S-phase entry. In contrast, by measuring RNA content in addition to DNA content, cells can be assigned to G₀ and cell-cycle subcompartments of G₁. Assessing phenotype at the same time as nucleic acid content allows determination of the cell-cycle status of subpopulations in mixed-cell preparations. This protocol describes an optimized method for combining dual-color cell-surfaceimmunofluorescent staining with staining for DNA-RNA, adapted for a basic dual-laser flow cytometer with blue (488-nm) and red (633- or 647-nm) excitation. DNA is stained at low pH inthe presence of saponin with 7-aminoactinomycin D (7-AAD), and RNA is stained with pyronin Y (PY). Both dyes are used at low concentration, and 7-AAD is exchanged with nonfluorescent actinomycin D to minimize fluorochrome-fluorochrome interactions, which can negatively affect detection of cell-surface antigen staining.

RELATED INFORMATION

This protocol was adapted from Schmid et al. (2000). An extensive description of flow cytometry principles, as well as various techniques for assessment of nucleic acid content by flow cytometry, can be found in Practical Flow Cytometry (Shapiro 2003).

MATERIALS

Reagents

  Actinomycin D (AD) stock solution (1 mg/mL)

  7-aminoactinomycin D (7-AAD) stock solution (1 mg/mL)

  Antibody staining solution

Human cells of interest, fresh or cultured (or immortalized human cell lines)

Monoclonal antibodies (mAb) directed against cell surface antigens of interest (biotinylated or labeled with an appropriate fluorochrome), and isotype-matched controls

The protocol described here uses one biotinylated mAb in combination with streptavidin-Alexa Fluor 488 and one mAb directly conjugated to allophycocyanin (APC) for combining dual-color immunofluorescence with DNA-RNA staining. See Troubleshooting and Discussion for further information on strategies for fluorochrome selection and staining.

  Nucleic acid staining solution (NASS) (pH 4.8)

 Phosphate-buffered saline (PBS, 1X, without Ca⁺⁺ and Mg⁺⁺)

  Pyronin Y(G) (PY) stock solution (1 mg/mL)

Streptavidin-Alexa Fluor 488

Equipment

Bucket with ice

Centrifuge

Flow cytometer with 488-nm blue excitation and 633-nm or 647-nm red excitation, and the appropriate filter sets for collecting emissions from Alexa Fluor 488, PY, 7-AAD, and APC

Incubator, preset to optimal temperature for cells of interest

METHOD

Cell-Surface Staining

• 1. Place 1 x 10⁶ PBS-washed cells into a tube, add 100 μL of antibody staining solution, and mix well. In addition, prepare cells to be stained with isotype-matched control antibodies, as well as cells to be stained with a single color, to be used as controls during flow cytometry. 
  The preparation of samples stained with isotype-matched control antibodies is necessary for determination of background staining. The preparation of single-color control samples is necessary to determine the fluorescence overlap of APC and Alexa Fluor 488 into other detectors on the flow cytometer and to set accurate fluorescence compensation. 
  
  

• 2. Label the cell-surface proteins of interest: 
  

  • i. Add appropriate amounts of a biotinylated mAb and an APC-labeled mAb to the cell sample. 
    
    

  • ii. Incubate the cells protected from light, using the optimal incubation temperature and time for the antibodies selected. 
    If the cell surface protein of interest is expressed at high levels, it may be possible to use a mAbthat is directly conjugated to Alexa Fluor 488, omitting the need for indirect staining using a biotinylated antibody. If this is the case, omit Steps 3 and 4, and proceed directly to Step 5. 
    

• 3. Wash the cells once by adding 2 mL of antibody staining solution and centrifuging at 250g for 5 min at 4°C. Discard the supernatant. 
  
  

• 4. To the cell pellet, add 100 μL of antibody staining solution containing an appropriate amount of streptavidin-Alexa Fluor 488. Incubate for 20 min at 4°C. 
  
  

• 5. Wash the cells once by adding 2 mL of antibody staining solution and centrifuging at 250g for 5 min at 4°C. Discard the supernatant.