Simultaneous Cell Surface, BrdU and DNA
Content Staining for Flow Cytometry
S. Rabin — 6/2003; v.2 6/2011
M. et al, Cytometry 1998)
- 1 x 106 cells cultured cells in suspension or
- 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,
- 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
- Label cells with by adding BrdU (final concentration 3
µg/mL) to the cell culture media for 1 hr.
- Purify cells on Ficoll gradient (clone 10 cells), or lightly
trypsinize fibroblasts to obtain a single cell suspension as
- Wash with PBS/FBS (by spinning at 1500 RPM and resuspending in
PBS/FBS) and transfer to a 1.5 mL eppendorf tube.
- Incubate in the dark with fluorochrome (PE)-conjugated cell
surface antibody (1:100 dilution in PBS/FBS) @ 4°C for 30 min.
- 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
- Warm PBS + Ca2+, Mg2+ to 37°C.
- Transfer cells to 15 mL conical tube and spin @ 1500 RPM for 2
- Discard supernatant, resuspend cell pellet in 100 µL
PBS/FBS, transfer to V-bottom 96 well plate, spin and wash 2X with
- Resuspend cells in 90 µL warm PBS + Ca/Mg and add 1 µL DNase I (10 mg/mL, 5 Kunitz U/µL). Incubate @ 37°C
for 30 min.
- 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.
- 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.
- 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.