1Department of Genetics, Harvard Medical School, 2 , Howard Hughes Medical Institute
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Perrimon, N., Zirin, J., Bai, J. Primary Cell Cultures from Drosophila Gastrula Embryos. J. Vis. Exp. (48), e2215, doi:10.3791/2215 (2011).
Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.
1. Preparation of fly cages
2. Collection of synchronous embryos
3. Collection and washing of embryos
4. Homogenization of embryos and plating of cells
5. Primary-cell RNAi for cells grown on a 384-well plate
6. Representative Results:
In optimal conditions, cells in the culture will look healthy with a round morphology. The culture will have little cell debris, and be 50~80% confluent after initial plating. These cells will undergo morphological changes after being cultured for an extended period of time. It usually takes about 5-8 days for cells in the culture to be fully differentiated at 25°C. Good quality primary cultures are usually judged by how well the cells-of-interest are differentiated in culture. For example, primary muscle cells often have a branch-like morphology and become multinucleated myotubes with stripe-like myofibrils consisting of a series of sarcomeres. Fully differentiated myotubes will usually spontaneously move in the culture. In contrast, differentiated primary neurons form clusters with their cell bodies, and connect with other clusters with their axon cables.
Figure 1. Primary cells undergo morphologic changes after being plated in the culture
(A) A bright field image of primary cells right after being plated in a well of a 384-well plate. The majority of cells are round and intact and well separated. (B) A bright field image of primary cell culture 20 hours after plating. Primary cells already form clusters (big arrowhead) and muscles with a branch-like morphology (long arrow) can be recognized at this stage. (C) A phase-contrast image of primary culture 5 days after plating. The culture looks much more advanced, with neuronal extensions (small arrows), neuronal clusters (big arrowheads) and muscles (medium-sized arrow).
Figure 2. Examples of primary cells treated with dsRNAs cultured in a 384-well plate
Primary cells were isolated from the embryos carrying Dmef2-Gal4, D42-Gal4, UAS-mito-GFP transgenes, in which Dmef2-Gal4 and D42-Gal4 drive expression of mito-GFP in muscles and motor neurons, respectively. The cells were treated with dsRNAs targeting either lacZ (A-C) or inflated(if) (D-F). Both primary muscles and neurons can be seen by GFP (A,C,D,F). The white arrows point to the neuronal extensions. Phalloidin staining of actin (arrowheads) reveals a branch-like muscle structure in the culture treated with lacZ dsRNAs (B and C), but round-up muscle morphology in the culture treated with if dsRNAs (E and F). In the merged images (C, F), muscles are stained yellow, but red negative shows neurons and their extensions (white arrows).
Figure 3. Confocal micrographs of primary muscles cultured on human vitronectin-coated cover glass slides
Primary muscles were immunofluorescently stained with anti-Drosophila Titin (KZ) (red in the merge) and with phalloidin for actin (green in the merge). The top panels are the wild-type control muscle and bottom ones are the sals(CG31374) RNAi muscle with shortened sarcomeres.
The developmental pattern of primary cultures of Drosophila cells can vary greatly, possibly due to the several variables during the primary cell preparation process. First of all, flies used for egg laying should be young (within a week old) and healthy (free of viral or bacterial infection). Unfertilized or infected embryos must be avoided, as they are useless and cells derived from those embryos cannot differentiate and will only survive for 1-2 days. Second, during the homogenization process, it is important not to overload the homogenizer with too many embryos. Overloading the homogenizer with too many embryos will cause an increase in the amount of debris and the number of dead cells, which will eventually compromise cell differentiation. Finally, cells should be plated at an appropriate density to ensure good differentiation of primary cells. We found that the differentiation of both neurons and muscle cells is dramatically reduced when primary cells are seeded initially at a too high density.
For the phenotypic analysis, 384-well plates are usually used to grow primary cells for screening or imaging analysis at a lower magnification. Images with a much higher magnification and better resolution can be achieved by growing cells on a cover-glass chamber slide, followed by imaging either live or stained cells using a confocal microscope. As most primary cells are adherent, the area for growing cells in the well of chamber slides should be used as a guideline for estimation of both the number of cells and the amount of medium required for each well. In addition, cover-glass chamber slides can be coated with extracellular matrix (ECM) proteins such as human vitronectin, laminin or collagen IV to allow differentiation of cells-of-interest. For example, primary muscles differentiate poorly on the non-coated cover-glass, but well on the ECM-coated cover-glass. Finally, in order to reduce autofluorescence emitted from culture medium, the regular culture medium such as Shield and Sang M3, should be replaced with a fly physiological saline buffer, such as HL6, right before imaging.
In principle, this protocol can be used for preparation of primary cells from flies with any genotypes, such as from homozygous viable and fertile mutant stocks, and from those whose embryos can be selected manually or by a sorter based on expression of GFP, or express tissue-specific Gal4, UAS-gene X genotypes. In combination with other experimentation methods, this protocol will allow a variety of questions to be addressed, especially those that are related to differentiated cells such as neurons and muscles, and are difficult to be tackled in cultured cell lines.
No conflicts of interest declared.
J.B. was supported by the Damon Runyon Cancer Research Foundation Fellowship DRG-1716-02. J.Z. was supported by NIH/NIGMS Fellowship F32 GM082174-02. N.P. is an investigator of the Howard Hughes Medical Institute.
|Shields and Sang M3 medium||Sigma-Aldrich||S3652|
|Fetal bovine serum||JRH Biosciences||12103-500M|
|Insulin from bovine pancreas||Sigma-Aldrich||I-6634|
|Large embryo collection cages||Genesee Scientific||59-101|
|#25 US standard testing sieve||VWR international||57334-454|
|#45 US standard testing sieve||VWR international||57334-462|
|#120 US standard testing sieve||VWR international||57334-474|
|Dounce tissue grinder, Wheaton 7 mL||VWR international||62400-620|
|Dounce tissue grinder,Kontes 40 mL||VWR international||KT885300-0040|
|Dounce tissue grinder,Kontes 40 mL||VWR international||KT885300-0100|
|Costar, 384-well plate||VWR international||29444-078|
|Lab-Tek II Chambered coverglass||Fisher Scientific||171080|