52
Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 4
Stem Cells from the Midgut
Midgut epithelium cells, columnar cells, and goblet cells differentiate from
stem cells of the midgut. Loeb and Hakim (1996) have developed a cell
system from the culture of midgut stem cells.
Materials
Equipment - Dissecting microscope and dissecting tray (see chapters 1-3)
- Centrifuge and centrifuge tubes
- Scissors
- Forceps
- Needles
- Sterile plastic pipette
- Pins
- Petri dishes
- 6-Well culture plates
Chemicals - Septisol, a detergent (Vestal Laboratories)
- p-Hydroxybenzoic acid methyl ester (Sigma)
Solutions - 0.1 % Sodium hypochlorite
- Ringer's solution
- Modified Grace's medium
Mix 200 ml Grace's medium, 14 ml calf serum, 0.2 g Yeastolate, 0.4 ml
Garamycin (0.5%),1.2 ml Vitamin premix (Roche Chemical Co.), and
antibiotic-antimycotic (Sigma).
- Fat body extract
Take fat body tissue from the abdomen of a new pupa of the tobacco
hornworm Manduca sexta.
Incubate fat body tissue from 30-40 animals in 2 ml medium/well of a
6-well culture plate for 24 h at 25°C.
Sonicate the fat body tissue in the incubation medium.
Apply two cycles of freeze-thawing.
Centrifuge the homogenate for 30 min at 1,600 g.
Take the clear fluid between the precipitate and the fatty overlayer.
Sterilize the clear fluid by passing it through a 0.22 11m filter.
Store the sterilized extract at -20°C in 500-111 aliquots.
Animals - Slipped-head stage of third instar larvae of M. sexta, Lymantria dispar
or Heliothis virescens or another suitable lepidopteran larvae
Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 4
Stem Cells from the Midgut
Midgut epithelium cells, columnar cells, and goblet cells differentiate from
stem cells of the midgut. Loeb and Hakim (1996) have developed a cell
system from the culture of midgut stem cells.
Materials
Equipment - Dissecting microscope and dissecting tray (see chapters 1-3)
- Centrifuge and centrifuge tubes
- Scissors
- Forceps
- Needles
- Sterile plastic pipette
- Pins
- Petri dishes
- 6-Well culture plates
Chemicals - Septisol, a detergent (Vestal Laboratories)
- p-Hydroxybenzoic acid methyl ester (Sigma)
Solutions - 0.1 % Sodium hypochlorite
- Ringer's solution
- Modified Grace's medium
Mix 200 ml Grace's medium, 14 ml calf serum, 0.2 g Yeastolate, 0.4 ml
Garamycin (0.5%),1.2 ml Vitamin premix (Roche Chemical Co.), and
antibiotic-antimycotic (Sigma).
- Fat body extract
Take fat body tissue from the abdomen of a new pupa of the tobacco
hornworm Manduca sexta.
Incubate fat body tissue from 30-40 animals in 2 ml medium/well of a
6-well culture plate for 24 h at 25°C.
Sonicate the fat body tissue in the incubation medium.
Apply two cycles of freeze-thawing.
Centrifuge the homogenate for 30 min at 1,600 g.
Take the clear fluid between the precipitate and the fatty overlayer.
Sterilize the clear fluid by passing it through a 0.22 11m filter.
Store the sterilized extract at -20°C in 500-111 aliquots.
Animals - Slipped-head stage of third instar larvae of M. sexta, Lymantria dispar
or Heliothis virescens or another suitable lepidopteran larvae