Dissection of the Midgut from an Adult Fly: A Method for Isolating the Digestive Tract

Abstract

Source: Tauc, H. M., et al. Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging. J. Vis. Exp. (2014).

In this video, we highlight a dissection of the adult Drosophila intestine and collection of midguts suitable for single-cell dissociation and FACS in order to isolate intestinal stem cells.

Protocol

This protocol is an excerpt from Tauc et al., Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging, J. Vis. Exp. (2014).

NOTE: If this protocol is used for the first time to isolate ISCs by FAC sorting, the following controls are mandatory in order to initially set the FACS parameters properly: Dissociated cells from wild type (e.g. w¹¹¹⁸) Drosophila midguts without Sytox. Dissociated cells from wild type (e.g. w¹¹¹⁸) Drosop....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Forceps: Dumont, Inox Biologie #5	Fine Science Tools	11252-20
SefarNitex 03-150um/38 (35 µm nylon mesh)	Sefar	3A03-0150-102-00
Falcon 5 ml Round Bottom Polystyrene Test Tube with Cell Strainer Snap Cap	Corning	352235
Polymax 1040	Heidolph	543-42210-00
Albumin from bovine serum (BSA)	Sigma	A4503-50G
0.5% Trypsin-EDTA	Invitrogen	15400-054	Trypsin obtained from a different company most likely has a different activity and the duration of the trypsin digest has to be adjusted accordingly.
SYTOX Blue Dead Cell Stain for flow cytometry	Life Technologies	S34857
RNAlater Stabilization Solution	Life Technologies	AM7023	Other solutions, e.g., Trizol can be used for subsequent RNA isolation
FACSAria II cell sorter	Becton Dickinson		Turn on one hour prior to sorting