Garland Cell assay
Background: Garland cells are large binucleate cells located as a ring of loosely attached single cells in front of the proventriculus (see cartoon above). These cells are extremely active in endocytosis and are popular as a tool to study endocytic trafficking in different mutant backgrounds (Narita K. et al). Since endocytosis inevitably leads to uptake of some fluid from the outside into the newly formed endocytic vesicle, the subsequent trafficking can be studied on the way to the lysosome by a simple pulse-chase experiment. Depending on how long the chase period is, subsequently later compartments of the endocytic pathway will be containing the endocytosed markers. For instance, the Garland cells were used to show the early endocytic defect of Hsc70 mutants (Chang HC. et al.) .
Protocol: Naturally, the animal with the mutation need to live to the larval stage to be able to easily locate and use the Garland cells for study of fluid phase endocytosis.
- Tear the animal in two parts to expose the midgut with the attached Garland cells (the image above was obtained using a normal stereo microscope used for dissection).
- Dump the tisse into a drop of labeling solution (about 50-100 ul) and leave it there for 5 minutes (pulse).
- Wash briefly in labeling free medium. Leave the tissue to incubate for the intended time (chase) until the marker has reached the compartment you are interested in. For instance, most of the endocytosed marker has reached the lysosome after 40 minutes of chase at 25 C (Lloyd TE. et al.).
- Visualize immediately in the microscope or stop the experiment by fixation in 4% formaldehyde in PBS buffer and proceed with conventional antibody staining.
Fluorescent probes (for confocal analysis): Use of fluorescently labeled probes is reliable and can be done in a matter of minutes in living cells. Use fluorescently labelled dextran (like Oregon green dextran, from Molecular probes ) and give a pulse of 5 minutes to visualize early endocytic compartments (endosomes). A counterstaining using Lysotracker ( Molecular probes ) is handy since it highlights the acidic (lysosomal) compartment in living cells and thus tells you wether the tracer is able to reach the lysosome (Lysotracker emits light only from acidic milieu and does not work well after fixation). After 40 minutes of chase the markers have reached the lysosomes (co-labelling with Lysotracker). Below an example is given where Garlnd cells have taken up OGD during a 5 min. pulse and 40 min chase countertainsed with lysotracker. Note the early endocytic vesicles close to the plasma membrane and the more centrally located acidic lysosomes.
Probes for Electron Microscopy (EM): The preferred classical probes for electron microscopy are BSA(Bovine Serum Albumin)-labelled gold or the enzyme Horse Raddish Peroxidase (HRP) (Lloyd TE. et al.). Pulse chase experiments using these markers allow the visulaisation of the result by Electron Microscopy allowing a complementary and detailed ultrastructural analysis of the process also observed by confocal analysis. The gold particles can be seen directly by EM after fixation. The cells containing internalised HRP are fixed, and HRP enzymatic activity visualized by conversion of the substrate DAB forming a dark perecipitate that can be seen by EM.
Labeling solution:
5-10 mg/ml Oregon Green Dextran in Schneider medium (or optionally SFM)
Wash and chase solution:
Schneider medium (or optionally SFM)
Fixative:
4% Formaldehyde in PBS for immunofluorescence (30 minutes fixation)
Lysotracker counterstaining:
Dump the live (unfixed) tissue in aPBS solution containing 1:200 dilution of Lysotracker for 2 minutes. Wash briefly (seconds), mount and vilualize immediately in the microscope.
PULSE CHASE
An experiment is initiated at a defined timepoint (pulse) and incubated incubated (chased) before the result is obtained. For analysis of endocytosis, this usually means providing a receptor ligand or a fluid phase tracer during the pulse and chase before looking at the trafficking of the internalised marker and/or its effect on the cell.