to BioTechniques free email alert service to receive content updates.

Immunostaining protocols for flow cytometric analysis of adherent cells
Ricardo B. deMedeiros, Ph.D. and Joy Aho, Ph.D.
Sponsored,vendor-submitted protocol    Sponsored by R&D Systems, Inc.    Published in November 2009 (p.11) DOI: 10.2144/000113280

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

The human embryonic stem cell line BG01V was stained with (A) APC-conjugated anti-human/mouse SSEA-4 (Cat. no. FAB1435A; surface antigen) and (B) PE-conjugated anti-human/mouse Oct-3/4 (Cat. no. IC1759P; intracellular antigen) monoclonal antibodies (filled histograms) or respective isotype control antibodies (open histograms), using the simultaneous fixation/permeabilization procedure described in the protocol.

1. Remove media from culture flask or plate and rinse cells with PBS or HBSS.

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.

More Information
 
Do you have question about this protocol? Would you like more information about this protocol?
Submit your questions here and Biotechniques will forward your question and contact information from our database to an expert at R&D Systems, Inc. who will contact you.
Yes, please forward my question, along with my name, title, company, and e-mail address to R&D Systems, Inc.
BioTechniques will forward your question to the company, who will contact you directly by email.
Request more information
 
Name Question
Job Title
Company
Email
Contact Information
Protocol Categories
rightcontent.jsp called otherwise condition rightcontent.jsp called 222222222222222222222
Search Protocols
Search our protocols for your specific technique.
Submit Your Protocols
BioTechniques is always looking for author-submitted protocols related to peer-reviewed papers, so submit your protocols today to our editorial team. Suppliers or advertisers interested in publishing their protocols should also submit.
Click here for more information