Simultaneous measurement of three-color immunofluorescence and DNA content

 

Materials

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

Newborn calf serum (NCS)

Sodium azide (NaAz)

Nucleic acid staining solution (NASS, phosphate-citrate buffer tablets, sodium chloride, sodium ethylene-diaminetetraacetic acid (EDTA), bovine serum albumin (BSA), all from Sigma), see recipe

Dimethylsulfoxide (DMSO)

Saponin (Sigma) 

7-amino-actinomycin D (7-AAD) stock solution, see recipe

Actinomycin D (C1) (AD, Roche Molecular Biosystems) stock solution, see recipe

Method

  1. Place 1 x 106 PBS-washed cells into a 12 x 75 mm tube and add 100 l of PBS supplemented with 2% NCS and 0.1% NaAz and mix well.

  2. For staining of cell surface antigen expression add appropriate amounts of biotinylated, phycoerythrin (PE)-labelled and allophycocyanin (APC)-labelled monoclonal antibodies (mAb) or of corresponding labelled isotypic control antibody and incubate the samples while protected from light for 15 min at 20oC - 25oC.

  3. Wash cells once with 2 ml of 1 X PBS by centrifugation at 250 x g for 5 min.

  4. Remove the supernatant and add 100 l of PBSAz containing 2 g of streptavidin Alexa Fluor488R and follow by incubation for 20 min at 4oC.  Wash cells once with 2 ml of PBSAz by centrifugation at 250 x g for 5 min at 4oC. 

  5.  Resuspend cells in 0.5 ml of NASS containing 0.02% of saponin and 10 g/ml of 7-AAD followed by incubation for 20 min at 20oC - 25oC protected from light.

  6. Then, spin cells down by centrifugation at 250 x g for 5 min.  Resuspend the cell pellet in 0.5 ml of NASS containing 0.02% of saponin and 10 g/ml of AD and place the mixture on ice protected from light for at least 10 min before acquisition on the flow cytometer.

Note: it is possible to replace cell surface staining with Alexa Fluor 488 which is not pH sensitive with staining using mAbs directly labelled with FITC and omit the second staining step with streptavidin; however, to restore the FITC fluorescence that is markedly diminished in NASS at pH 4.8, it is  necessary to resuspend cells after the spin in step 6 in 1 X PBS at pH 7.2 containing 0.02% of saponin and 10 g/ml of AD and place them on ice for at least 10 min before acquisition on the flow cytometer.

Preparation of solutions:

7-AAD stock solution (1mg/ml): dissolve 1 mg of 7-AAD powder first in 50 l of DMSO, then add 950 l of 1 X PBS; keep at 4oC protected from light.

Nucleic acid staining solution (NASS, pH 4.8): 0.15 M NaCl in 0.1 M phosphate-citrate buffer containing 5 mM sodium EDTA and 0.5% BSA fraction V

Dissolve 2 tablets of phosphate-citrate buffer in 100 ml of distilled H20 to make a 0.1 M solution.

Add 0.18 g of disodium EDTA to a final concentration of 5 mM.

Add 0.9 g of  NaCl to a final concentration of 0.15 M.

Add 0.5 g of  BSA to a final concentration of 0.5%.

Keep at 4oC.

Actinomycin D (AD) stock solution (1mg/ml): dissolve 1 mg of AD powder first in 50 l of DMSO, then add 950 l of 1 X PBS, keep at 4oC protected from light.

Reference:

Schmid I, Cole SW, Zack JA, Giorgi JV.  Measurement of lymphocyte subset proliferation by three-color immunofluorescence and DNA flow cytometry. J Immunol Meth. 235:121-131, 2000.