Drosophila cell line protocols for modENCODE

In what follows, we assume that the user has some basic tissue culture experience. For general procedures, we recommend that modENCODE users read the protocol for general Drosophila cell line maintenance. If you need more detailed advice than you find in this protocol plus the protocols on the DGRC web-site, contact the DGRC for help; all cell line queries addressed to dgrc@indiana.edu will be read by Lucy Cherbas, and answered either by her or by another qualified DGRC cell line staff member.

Acquiring the lines

The 4 cell lines on which modENCODE Drosophila projects will concentrate are:

For detailed information about each line, see the protocols for the individual lines.

For each of these lines, the DGRC has designated a lot of frozen ampoules for modENCODE, in order that all modENCODE projects will receive identical cells. Orders must be placed through the DGRC web-site. When you place an order with the DGRC for cells to be used on the modENCODE project, please send an e-mail at the same time to dgrc@indiana.edu, stating that the cells in this order are for modENCODE. That will ensure that the DGRC staff sends you cells from the modENCODE-designated frozen stock.

The DGRC ships cells as frozen ampoules on dry ice. Follow the instructions for thawing.

Note: Please thaw the cells as soon as possible upon receipt; the cells may be damaged by temperature fluctuations during shipment, and putting them back at -80° or into liquid nitrogen may only make things worse.

With the exception of ML-DmBG3-c2 (for which an MTA is required - see below), all of these lines may be freely distributed. For the sake of consistency, each group should start with cells from the designated modENCODE lot at the DGRC. These cells may be distributed to others within modENCODE, but we recommend that this not be done by shipping flasks of growing cultures; there is sufficient damage to cells during shipment of a growing culture that the properties of the cells may change.

In order to ensure that the cells do not change significantly during the period they are grown, the culture should be re-started from a frozen stock at regular intervals (see individual cell line descriptions for recommended times of continuous growth). You have two choices for doing this: You may order another ampoule of cells from the DGRC, or you may use a stock of frozen cells which you make yourself as soon as possible after the original cells shipped by the DGRC have recovered and are growing well (see https://dgrc.bio.indiana.edu/include/file/FreezingCells.pdf for freezing protocol).

Note: We are in the process of posting a revision of the freezing protocol on the DGRC web-site; the only change is that we now recommend using 20% FCS rather than 10% FCS in the freezing medium. Be sure to use the higher serum concentration, which seems to give better preservation of the cells during prolonged storage.

General growth conditions

All of the lines should be grown in 100 mm tissue-culture grade Petri plates (Corning #430167) at 25° in a humidified chamber; use 10 ml of culture in each plate. Use a 25° incubator without added CO2.

Note: We have noticed slight changes in properties of some of the lines grown in plasticware from different manufacturers; therefore, in order to maximize the similarity of cells used in different labs, all modENCODE experiments should be done using cultures grown in Corning plates. There are also differences between growth in Petri plates and in T-flasks; therefore use only Petri plates for experiments, and restrict the use of T flasks to thawing cells and carrying cells from one lab to another. Do NOT use spinner flasks to grow cells; several of the lines will grow well in spinner flasks, but cells in spinner flasks are quite different from the same cells growing in plates.
Note: The simplest form of humidified chamber is a sealed plastic food-storage container (e.g. Tupperware or Rubbermaid). You do not need to add water to the container; the tissue culture plates generate enough humidity to keep the plates from drying out. If you prefer, you can put a tray of water in the incubator to keep the humidity up, but this is more messy and more likely to grow mold.

Medium

Most of the cell lines used in the Drosophila modENCODE project are grown in Schneider's medium, which is purchased as a liquid medium, ready to use, from Sigma/Aldrich (cat. no. S0146). Cl.8 cells are grown in M3 medium, which is also purchased as a liquid medium, ready to use, from Sigma/Aldrich (cat. no. S3652). Both media are stored at 4°; please observe the expiration date on the label.

Before use, add heat-inactivated fetal calf serum to the final concentration recommended for each line. Some cell lines require additional additives; see instructions for the individual lines.

Note: We do not recommend buying Schneider's medium from another supplier, because medium from different companies may be slightly different in pH or in other properties. Similarly, we do not recommend buying powdered medium from Sigma and making it up yourself, because that risks variations in water and pH.
Note: We recommend not using antibiotics, which may have minor effects on the properties of the cells. The use of a sterile hood (laminar flow or BioSafety), with standard sterile technique, is normally sufficient to prevent bacterial and fungal contamination. Occasionally an individual has a sufficiently high level of skin flora that these precautions are not enough; in this case, wearing lab gloves should solve the problem.
Note: Schneider's medium is not the medium recommended on the DGRC web-site. We have chosen to use Schneider's medium for the modENCODE project because all 4 of the cell lines grow well in it, and it is commercially available, ready to use, thereby minimizing variations in preparation between labs.

Fetal calf serum

Fetal calf serum will be purchased by the Cherbas lab (all from a single lot: HyClone cat. no. SH30070.03, lot ASD29137). On request, bottles will be thawed and heat-treated by the Cherbas lab (56°, 60 min), and shipped by overnight Fedex to laboratories in the modENCODE consortium; the recipient laboratory will be expected to provide a Fedex account number to be used for the shipment, and will be billed for the costs to the Cherbas lab ($310 per 500 ml bottle of serum, plus approximately $10 per shipment for shipping materials). The heat-treated serum can be stored at 4° for up to several months. While this document has been preserved for reference, the Cherbas lab no longer provides fetal calf serum.


Citing the DGRC

When publishing experiments using materials obtained from the DGRC please follow the citation guidelines on the material's stock page, including citing the Drosophila Genomics Resource Center, supported by NIH grant 2P40OD010949, in the acknowledgments. Your cooperation helps us when we need to renew our grant as well as the researchers that donate materials to the DGRC.