5 Insecta (Lepidoptera)
61
Alternative Procedure
1. The following procedure is the same up to step 2 of the preceding
procedure.
1. Drop the blood into a centrifuge tube containing Carlson's solution.
Blood
2. Mix the blood and the solution by pipetting.
collection
3. Collect sufficient blood from several insects by repeating steps 2 and
3 (use many insects, especially if only a small amount of blood can be
obtained from a single insect).
1. Centrifuge at 150 g for 5 min.
Culture
2. Discard the supernatant.
set-up
3. Disperse the cell pellet into Carlson's solution.
4. Repeat steps 1-3 twice more to wash and remove the phenol oxidase.
5. Suspend the final cell pellet in culture medium instead of Carlson's
solution.
6. Transfer the cell suspension into a culture vessel.
Results
Plasmatocytes stretch their cytoplasm thinly, like a membrane, and take
on a very flat form. Granular hemocytes develop slender cytoplasmic
projections. Prohemocytes maintain their smooth surface (Fig. 12).
Spherule cells and oenocytoids do not change their shape, but gradually
deteriorate, and, ultimately, die. Often, various types of cells will aggregate to form cell masses, and from these aggregates cells will come out
into their surroundings (Fig. 12). Sometimes the culture medium becomes
dark because of phenol oxidase that has remained on or in the cells. When
melanization occurs, wash the culture several times with Carlson's solution, and continue the culture with fresh culture medium.
Usually,hemocytes in primary cultures are short-lived, e.g., 1 week or 10
days. However, sometimes cells begin to divide. In most cases, multiplication starts with the prohemocytes. Plasmatocytes and granular
hemocytes may also divide. When cells fill the culture vessel, make a
subculture (see chapter 4, subprotocoI4). By repeating the subculture, it
is possible to obtain a continuously growing cell line from hemocyte cultures. Several hemocyte cell lines have been established (Chao and Ball,
1971; Miltenburger et aI., 1977; Mitsuhashi and Shozawa, 1985).
Subprotocol 9
Hemocytopoietic Organs
In Lepidoptera, hemocytopoietic organs (HPOs) exist attached to wing
discs in meso- and metathoraces. The shape of the organ is ring like
61
Alternative Procedure
1. The following procedure is the same up to step 2 of the preceding
procedure.
1. Drop the blood into a centrifuge tube containing Carlson's solution.
Blood
2. Mix the blood and the solution by pipetting.
collection
3. Collect sufficient blood from several insects by repeating steps 2 and
3 (use many insects, especially if only a small amount of blood can be
obtained from a single insect).
1. Centrifuge at 150 g for 5 min.
Culture
2. Discard the supernatant.
set-up
3. Disperse the cell pellet into Carlson's solution.
4. Repeat steps 1-3 twice more to wash and remove the phenol oxidase.
5. Suspend the final cell pellet in culture medium instead of Carlson's
solution.
6. Transfer the cell suspension into a culture vessel.
Results
Plasmatocytes stretch their cytoplasm thinly, like a membrane, and take
on a very flat form. Granular hemocytes develop slender cytoplasmic
projections. Prohemocytes maintain their smooth surface (Fig. 12).
Spherule cells and oenocytoids do not change their shape, but gradually
deteriorate, and, ultimately, die. Often, various types of cells will aggregate to form cell masses, and from these aggregates cells will come out
into their surroundings (Fig. 12). Sometimes the culture medium becomes
dark because of phenol oxidase that has remained on or in the cells. When
melanization occurs, wash the culture several times with Carlson's solution, and continue the culture with fresh culture medium.
Usually,hemocytes in primary cultures are short-lived, e.g., 1 week or 10
days. However, sometimes cells begin to divide. In most cases, multiplication starts with the prohemocytes. Plasmatocytes and granular
hemocytes may also divide. When cells fill the culture vessel, make a
subculture (see chapter 4, subprotocoI4). By repeating the subculture, it
is possible to obtain a continuously growing cell line from hemocyte cultures. Several hemocyte cell lines have been established (Chao and Ball,
1971; Miltenburger et aI., 1977; Mitsuhashi and Shozawa, 1985).
Subprotocol 9
Hemocytopoietic Organs
In Lepidoptera, hemocytopoietic organs (HPOs) exist attached to wing
discs in meso- and metathoraces. The shape of the organ is ring like