Abstract

Flow cytometry is a powerful technique for assessing the expression of multiple proteins simultaneously in individual cells within a heterogeneous population. This technique is most commonly used with non-adherent cells, but it can also be an effective tool for analyzing protein expression in adherent cells. The following immunostaining protocol outlines the steps required for detecting surface or intracellular antigens in adherent cells by flow cytometry. Data is presented to show the staining of SSEA-4 (surface) and Oct-3/4 (intracellular) in BG01V embryonic stem cells using this protocol.

Materials

PBS/EDTA (PBS, 2 g/L EDTA, Cat. no. E9884; Sigma, St. Louis, MO, USA)

Trypsin solution [cell media with 5 g/L Trypsin (Cat. no. T1426; Sigma, St. Louis, MO, USA), and 2 g/L EDTA] or Gibco 0.05% Trypsin-EDTA 1× (Cat. no. 25200-056; Invitrogen, Carlsbad, CA, USA), or Trypsin EDTA 1× (Cat. no. 9341; Irvine Scientific, Santa Ana, CA, USA), or other cell detachment solutions [Accutase™ (Cat. no. SCR005; Millipore, Billerica, MA, USA), Detachin™ (Cat. no. T100100; Genlantis, San Diego, CA, USA), or Cellstripper™ (Cat. no, 25-056-CI; MediaTech, Manassas, VA, USA)]

Flow cytometry staining buffer (Cat. no. FC001; R&D Systems)

Flow cytometry fixation buffer (Cat. no. FC004; R&D Systems)

Flow cytometry permeabilization/wash buffer I (Cat. no. FC005; R&D Systems)

Flow cytometry fixation/permeabilization buffer I (Cat. no. FC007; R&D Systems)

Adherent cell lines: HeLa, MCF-7, HEK293, HUVEC, RAW264, BG01V (licensed from BresaGen, Inc., Athens, GA, USA), etc.

Antibodies: APC-conjugated anti-human/mouse SSEA-4 (Cat. no. FAB1435A; R&D Systems) and PE-conjugated anti-human/mouse Oct-3/4 (Cat. no. IC1759P; R&D Systems)

Methods

A. For surface antigens

2. To remove adherent cells:

a. Add Trypsin solution (a volume large enough to cover the bottom of the flask) and keep at room temperature for 10 min or less (incubation time should be optimized by the investigator). Pipet up and down to gently remove the adherent cells. (Note: Trypsinization may significantly remove or decrease some surface antigens. This should be determined by the investigator prior to the experiment.)

b. Alternatively, use a scraper or pipet the cells up and down gently with PBS/EDTA solution.

c. Some cell lines may work better with a gentler cell detachment solution (several are listed above). When using these commercial solutions, follow the manufacturer’s instructions.

3. Following cell detachment, check under a light microscope to make sure the cells are not in clumps. Pipet up and down three times to break up any clumps.

4. Centrifuge and resuspend the cells at ~1 × 10⁶ cells/100 L flow cytometry staining buffer in a 5 mL flow cytometry tube.

5. Add antibody to the cells (at optimal concentration) and incubate for 30 min (2–8º C).

6. Remove excess antibody by washing the cells with 2 mL flow cytometry staining buffer and resuspend the final pellet in 200–400 L of the same buffer for flow cytometric analysis.

B. For intracellular antigens (with fixation followed by permeabilization)

1. Repeat steps 1–3 from section A.

2. Wash cells twice with PBS or HBSS.

3. Resuspend the cells in 500 L flow cytometry fixation buffer and incubate at room temperature for 10 min. Vortex cells intermittently to maintain a single-cell suspension.

4. Wash cells twice with PBS or HBSS and resuspend in 100–200 L flow cytometry permeabilization/wash buffer.

5. Add antibody to the cells (at optimal concentration) and incubate for 30 min (2–8º C).

6. Remove excess antibody by washing the cells with 2 mL flow cytometry permeabilization/wash buffer. Resuspend the final pellet in 200–400 L flow cytometry staining buffer for flow cytometric analysis.

C. For simultaneous fixation/permeabilization staining

1.Repeat steps 1–3 from section A.

2.Wash cells twice with PBS or HBSS.

3.Resuspend the cells in 500 L flow cytometry fixation/permeabilization buffer and incubate at 2–8º C for 30 min. Vortex cells intermittently to maintain a single-cell suspension.

4.Centrifuge the cells and resuspend in 100–200 L flow cytometry permeabilization/wash buffer.

5.Add antibody to the cells (at optimal concentration) and incubate for 30 min (2–8º C).

6.Remove excess antibody by washing the cells with 2 mL flow cytometry permeabilization/wash buffer. Resuspend the final pellet in 200–400 L flow cytometry staining buffer for flow cytometric analysis.