Simultaneous Cell Surface, BrdU and DNA Content Staining for Flow Cytometry

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S. Rabin — 6/2003

(Ref: Holm, M. et al, Cytometry 1998)

Materials

1 x 10⁶ cells cultured cells in suspension or monolayer

BrdU 3 mg/mL (1,000x stock) store frozen

PBS/FBS (PBS with 1% FBS)

Fixative: 1% paraformaldehyde + 0.05% v/v NonidetP40 in PBS

PBS Ca2+/Mg2+ (PBS with 0.1 g/L CaCl₂, 0.1 g/L MgCl₂*6H₂O, Gibco)

V-bottom 96 well plates

Cell surface antibody (e.g. PE conjugated Pharmingen antibodies diluted 1:100 in PBS/FBS)

DNAse I (Sigma, catalogue no. DN-25)

FITC-conjugated anti-BrdU (Pharmingen)

DAPI (10 µg/mL in PBS/FBS) (alternatively P.I. or 7-AAD may be used at this same concentration)

Flow cytometry tubes

Procedure

1. Label cells with by adding BrdU (final concentration 3 µg/mL) to the cell culture media for 1 hr.
2. Purify cells on Ficoll gradient (clone 10 cells), or lightly trypsinize fibroblasts to obtain a single cell suspension as appropriate.
3. Wash with PBS/FBS (by spinning at 1500 RPM and resuspending in PBS/FBS) and transfer to a 1.5 mL eppendorf tube.
4. Incubate in the dark with fluorochrome (PE)-conjugated cell surface antibody (1:100 dilution in PBS/FBS) @ 4°C for 30 min.
5. Wash cells 3X with PBS/FBS. Loosen pellet and then resuspend cells in 1mL Fixative in a 1.5 mL Eppendorf tube and rock o/n in the dark @ 4°C.

NEXT DAY

1. Warm PBS + Ca2+, Mg2+ to 37°C.
2. Transfer cells to 15 mL conical tube and spin @ 1500 RPM for 2 min.
3. Discard supernatant, resuspend cell pellet in 100 µL PBS/FBS, transfer to V-bottom 96 well plate, spin and wash 2X with PBS/FBS.
4. Resuspend cells in 90 µL warm PBS + Ca/Mg and add 10 µL DNase I (10 mg/mL, 5 Kunitz U/µL). Incubate @ 37°C for 30 min.
5. Wash 1X with PBS/FBS, resuspend in 80 µL PBS/FBS, add 20 µL FITC-conjugated anti-BrdU antibody. Incubate in the dark @ 4°C for 1 hr.
6. Wash 2X with PBS/FBS. Resuspend cells in 0.2 mL PBS/FBS + 10 µg/mL DAPI and transfer to flow cytometry tubes. (Alternatively, PI may be used.) Incubate in the dark @ 4°C for 30 min.
7. Do analysis with the LSR machine if DAPI is used for DNA content analysis. FACS or Calibur machines may be used if PI is used for DNA content. (See Setup and Use of the Flow Cytometer). Collect data with with the machine on Low or Medium flow rates to avoid compression of the DNA content profile.