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General Stem Cell Protocols


Alkaline Phosphatase Detection Kit

Embryonic stem (ES) cells are totipotent cells derived from the inner cell mass (ICM) of preimplantation mammalian embryos and are capable of unlimited, undifferentiated proliferation in vitro (Evans, 1981, Nature; Martin, 1981, PNAS; Morrison, 1987, Cell) Undifferentiated murine ES cells can be maintained in vitro for extended periods in media containing the cytokine, leukemia-inhibitory factor (LIF) or Millipore’s proprietary ES cell culture reagent, ESGRO mLIF Medium Supplement (Smith, 1988, Nature; Williams, 1988, Nature). The undifferentiated state of ES cells can be characterized by high level of expression of Alkaline Phosphatase (AP) (Pease, 1990, Dev. Biol.) the expression of surface markers including SSEA and TRA antigens and the transcription factor Oct-4.

This protocol describes the use of the Alkaline Phosphatase Detection Kit (Millipore cat. no. SCR004) for the phenotypic assessment of ES cell differentiation. Available separately from Millipore are the monoclonal antibodies TRA-2-49 (Millipore cat. no. MAB4349) and TRA-2-54 (Millipore cat. no. MAB4354), which permit the detection of Liver / Bone / Kidney isozyme of AP as expressed by ES cells. When used in flow cytometry, these reagents permit a quantitative assessment of AP expression by ES cells (Draper, 2002, J. Anat.).


Related Products

The following related products are available from Millipore as separate items:

  • ES Cell Characterization Kit — Millipore cat. no. SCR001
  • ES Marker Sample Kit — Millipore cat. no. SCR002
  • ES Cell 3D Collagen Culture Kit — Millipore cat. no. SCR003
  • SSEA-1 Monoclonal Antibody, purified 100µg — Millipore cat. no. MAB4301
  • SSEA-3 Monoclonal Antibody, purified 100µg — Millipore cat. no. MAB4303
  • SSEA-4 Monoclonal Antibody, purified 100µg — Millipore cat. no. MAB4304
  • TRA-1-60 Monoclonal Antibody, purified 100µg — Millipore cat. no. MAB4360
  • TRA-1-81 Monoclonal Antibody, purified 100µg — Millipore cat. no. MAB4381
  • TRA-2-49 Monoclonal Antibody, purified 100 µg — Millipore cat. no. MAB4349
  • TRA-2-54 Monoclonal Antibody, purified 100 µg — Millipore cat. no. MAB4354
  • Murine LIF 5 µg — Millipore cat. no. LIF2005, 10 µg — Millipore cat. no. LIF2010
  • ESGRO mLIF Medium Supplement, 1 x 106 units — Millipore cat. no. ESG1106, 1 x 107 units — Millipore cat. no. ESG1107

Kit Components

  • Fast Red Violet solution (0.8 g/L stock; 2x 15/mL bottles) — Millipore cat. no. 90239
  • Napthol AS-Bl phosphate solution (4mg/mL) in AMPD buffer (2 mol/L, pH 9.5) (15 mL bottle) - Millipore cat. no. 90234

Required Materials

  • Fixative (e.g. 4% Paraformaldehyde)
  • 1x Rinse Buffer (e.g. TBST: 20 mM Tris-HCI, pH 7.4, O.15M NaCl 0.05% Tween-20)
  • Hematoxylin (optional)
  • Microscope

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Protocol


Preparation of Reagents

Naphthol/Fast Red Violet Solution: Mix Fast Red Violet (FRV) with Naphthol AS-BI phosphate solution and water in a 2:1:1 ratio (FRV: Naphthol: water).


Staining Protocol
  1. Culture ES cells for five days prior to analyzing AP activity, at low to medium density.

    Note: This time-period is critical if activity levels of AP needs to be observed. According to our findings, five days of culturing are optimal for good AP stain visualization.

  2. On day five, aspirate media and fix ES or EC cells with a fixative (e.g. 4% Paraformaldehyde in PBS) for 1–2 minutes.

    Note: Do not overfix. Fixing cells longer than 2 minutes will result in the inactivation of alkaline phosphatase.

  3. Aspirate fixative and rinse with 1x Rinse Buffer. DO NOT allow wells to dry.
  4. Prepare reagents for Alkaline Phosphatase staining as described in “Preparation of Reagents” section.
  5. Add enough stain solution to cover each well (e.g. 0.5 mL for a well of a 24-well plate). Incubate in dark at room temperature for 15 minutes.
  6. Aspirate staining solution and rinse wells with 1x Rinse Buffer. Cover cells with 1x PBS to prevent drying and then count the number of colonies expressing AP (red stem cell colonies), versus the number of differentiated colonies (colorless).
  7. AP staining criteria: Greater than 90% of colonies should remain undifferentiated and express alkaline phosphatase in the well containing 103 Units of LIF. P value shall be = 0.05.

Staining Results with Alkaline Phosphatase Detection Kit

Alkaline Phosphatase Staining of ES Cells. High magnification revealed (A) Undifferentiated ES cells (moue MBL.5 cell line) — cultured for five days in media containing Millipore's mUF or ESGRO supplement. A concentration of 10³ Units/mL is used for inhibition of differentiation. (B) Differentiated ES cells — cultured at low-medium density for three days in media without any mUF or ESGRO supplement. (C) Differentiated ES cells — cultured at low-medium density for six days in media without any mUF or ESGRO supplement.


References

  1. Evans M, Kaufman M. Nature 1981; 292:154.
  2. Martin G. Proc Natl Acad Sci USA 1981; 78:7634.
  3. Morrison SJ, Shah NM, Anderson DJ. Cell 1997; 88:287–298.
  4. Smith AG, Heath JK et al. Nature 1988; 336:688–690.
  5. Williams RL, Hilton DJ et al. Nature 1988; 366:684–687.
  6. Pease S, Braghetta P et al. Develop Biol 1990; 11:344.

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