Exercise 13.8 - Prestalk vs Prespore Effect
Figure 13.7 Prestalk/prespore immunofluoresence
- Agar cultures of D. discoideum
- 20 mM Na-K Phosphate buffer, pH 7.0
- 20 mM Na-K Phosphate buffer plus 20 mM EDTA, pH 7.0
- Hypodermic syringe with 16 gauge needle
- Nylon mesh
- 70% v/v Percoll in 20 mM EDTA, 5 mM MES, pH 7.0
- Preparative centrifuge, rotor and tubes
- Collect migrating slugs by washing them from the agar with
cold 20 mM Na-K Phosphate Buffer, pH 7.0. A bent glass rod may be
used to assist in dislodging the slugs.
- Wash the slugs once with the same buffer and resuspend in 20
mM Na-K Phosphate Buffer containing 20 mM EDTA.
- Pipette the slugs up and down through a 16 gauge needle for
about 5 minutes to dissociate the cells. 29
- Pass the suspension of cells through a fine nylon mesh to
remove clumps, wash with the EDTA buffer and resuspend in 2-3 ml
of fresh EDTA buffer. Collect the cells by centrifuging for 2
minutes at 300 xg. Resuspend the cells to make a final
concentration of 4 X 10
cells per ml of EDTA buffer.
- Form two Percoll gradients by placing 12 ml of Percoll into
two centrifuge tubes and centrifuging at 21,000 xg for 40 minutes
at 4° C.
- Layer 250 µl of the cell suspension onto a preformed
Percoll gradient. Either distribute the cells between the two
gradients, or use one tube as a balance in the centrifuge.
- Centrifuge the amoeba in the preformed Percoll gradient at
13,000 xg at 15° C for 3 minutes.
- Collect and identify the bands of cells. Wash the cells free
of Percoll with EDTA buffer.
Once separated on Percoll, the cells can be analyzed for any
number of activities. One of the principal analyses is for the
production of specific gene products which would indicate DNA
control of differentiation. The effect of differential gene
activity can be studied by extracting RNA from the prestalk and
prespore cells with subsequent analysis of the RNA.
Since the ultimate gene product is a protein, several
investigators have identified and isolated proteins
characteristic of prestalk and prespore differentiation. By
producing antibodies to those proteins, immunofluorescent
techniques may be applied to the detection of early
differentiation. Figure 13.7
demonstrates such an analysis applied to prestalk/prespore cells.
to Table of Contents
Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org