Tpo Il3 Il6 Il11

Fig. 2. Examples of murine hematopoietic colonies. (A) Megakaryocyte colony in collagen stained with acetylcholinesterase. (B) Primitive erythroid colony composed of large red cells. (C) Macrophage colony composed of large clear cells. (D) Definitive erythroid colony (derived from a BFU-E) composed of small red cells. See color insert following p. 80.

Fig. 2. Examples of murine hematopoietic colonies. (A) Megakaryocyte colony in collagen stained with acetylcholinesterase. (B) Primitive erythroid colony composed of large red cells. (C) Macrophage colony composed of large clear cells. (D) Definitive erythroid colony (derived from a BFU-E) composed of small red cells. See color insert following p. 80.

4. Carefully place a filter card over the spacer. The card will wet completely over 5 to 10 min Do not adjust the filter card once it is placed on the slide.

5. Remove the filter card, leaving the polypropylene spacer on the slide. Place the slides horizontally in the cold acetone and fix for 5 min on ice. The polypropylene spacer will float off in the acetone.

6. Remove the slides from the acetone and air dry. The dehydrated slides can be stained immediately or stored at 4°C or -20°C in the dark up to 1 mo.

3.5.3. Identification of Megakaryocyte Colonies by Acetylcholinesterase Stainin

Acetylcholinesterase is present in multiple hematopoietic lineages, however it is highly expressed in mature megakaryocytes. Thus, acetylcholinesterase staining provides a means of identifying megakaryocytes in collagen culture (11), obviating reliance on morphology alone. We have applied acetylcholinesterase staining to methylcellu-lose cultures to identify megakaryocyte colonies (see Subheading 3.6.3.).

1. Dissolve 10 mg of acetylthiocholiniodide in 15 mL of 0.1 M sodium phosphate buffer.

2. To the acetylthiocholiniodide, add in the following order with constant stirring:

1 mL of 0.1 M sodium citrate

2 mL of 30 mM copper sulfate

2 mL of 5 mM potassium ferricyanide solution The total volume of 20 mL is sufficient to stain 10 to 15 slides.

3. Allow slides to come to room temperature before staining. Pipet 0.75 to 1 mL of the stain onto the dehydrated collagen, enough to cover the dried collagen. Incubate the slides at room temperature in a humid chamber for 3.5 h to detect large mature megakaryocytes, and up to 5 h to detect immature megakaryocytes.

4. Pour off staining solution and fix the slides horizontally in 95% ethanol for 10 min.

5. Rinse slides with lukewarm tap water and air dry.

6. Megakaryocytes colonies range in size from 3 to 50 cells and stain orange-brown to brown-black depending upon the amount of acetylcholinesterase activity in the cell. A typical megakaryocyte colony derived from the E8.5 yolk sac and stained with acetylcholinesterase is shown in Fig. 2A.

3.6. Culture of Erythroid, Myeloid, and Megakaryocyte Colony-Forming Cells in Methylcellulose

Unilineage, bilineage, and multilineage hematopoietic progenitors can be identified by the colonies they generate when suspended in methylcellulose. Serum lots and cytokine combinations greatly influence the colony types that can be generated.

3.6.1. Preparation of Methylcellulose Cultures

Methylcellulose is very viscous, requiring a 16-gage needle for aliquotting. Each batch of methylcellulose must be tested to determine the final concentration that optimizes colony growth. We have found that the final methylcellulose concentration typically falls between 55 to 65% of the starting solution. The following growth factors, serum, and other reagents are added to the methylcellulose.

Final concentration

Methylcellulose 55-65%

Serum (PDS or GemCell) 10%

Protein-free hybridoma medium 5%

Glutamine (100X) 1X

1X IMDM to final volume

3.6.2. Plating Methylcellulose

1. Aliquot 1.4 mL of the methylcellulose mix per dish into a 5-mL tube and add 0.1 mL of cells at the appropriate concentration.

2. Vortex vigorously and allow the bubbles to surface for 15 to 20 min at room temperature.

3. Use a 3-mL syringe with a 16-gage needle to plate 1 mL into each 35 x 10-mm culture dish.

4. Swirl the dish to distribute the methylcellulose evenly.

5. Plate enough cells to count between 50 and 500 colonies per 35-mm dish (see Note 7).

6. Cultures are incubated at 37°C, 5% CO2, room air for 2 to 16 d as needed to quantify colony numbers.

3.6.3. Colony Identification

The identification of colonies in semisolid media is often difficult. Colony morphology is dependent on the concentration of methylcellulose, the combination of growth factors and type of serum used, the length of time in culture, and the combination of lineages present in the colony.

1. Colony kinetics. Hematopoietic progenitors give rise to mature colonies over different periods of time, ranging from as short as 2 d for CFU-E to as long as 16 d for mast cell progenitors. The approximate times for optimal growth of the various hematopoietic progenitors are listed in Table 1. This wide range in colony timing necessitates examination of the dishes at multiple time-points to enumerate the various colony types.

2. Colony morphology. Each colony type has a distinct morphology and range of sizes. Myeloid and megakaryocyte colonies consist of clear cells, while erythroid colonies turn red (Fig. 2). Primitive erythroid colonies (e.g., EryP-CFC) are relatively small, bright cherry red colonies (Fig. 2B), whereas definitive erythroid (e.g. BFU-E) are larger and

Table 1

Murine Hematopoietic Progenitors That Give Rise to Distinct Colony Types

Table 1

Murine Hematopoietic Progenitors That Give Rise to Distinct Colony Types

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