Cell culture (fly)

Below we describe basic media, growth conditions, etc. for Drosophila cell culture.

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Informative reference materials include Drosophila Cells in Culture (Second Edition), edited by G. Echalier, N. Perrimon and S. Mohr.
 

 

Outline:

1. Medium
     A. S2, S2C, S2*, S2R+ (and all derivatives), S3, Kc(167),DL1, & DL2 cells
     B. ML-DmBG2 & ML-DmBG6 cells
     C. Clone8 cells
     D. Selective media
     E. Heat inactivating FBS
2. Insulin Stock
3. Fly Extract
4. Growth Conditions
5. Maintenance
     A. Semi-adherent cell lines
     B. Adherent cell lines
     C. Passing cells in flasks
     D. Passing cells in wells
     E. Expanding S2R+ cells and derivatives
6. Freezing
7. Thawing
8. Counting

 

1. MEDIUM

A. S2, S2C, S2*, S2R+ (and derivatives), S3, Kc(167), DL1, & DL2 cells:

Schneider's/ 10% FBS/ PS
450 mL Schneider's medium (Gibco #11720-034)
(pour off 50 mL into Falcon to save as serum-free)
***[S2R+ and Kc cells will not grow in Schneider's medium from Sigma]
50 mL Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
An alternative Heat Inactivated FBS, tested with S2, S2*, S2R+ & Kc(167), is HyClone (#SH30070)
5 mL 1:100 Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)
Sterile filter (0.2 µm) Store at 4°C - do not freeze. Cloudiness may appear after a few weeks. If cloudiness appear, discard the media and do not use on cells.

B. ML-DmBG2 & ML-DmBG6 cells:

M3/10% FBS/PS/Insulin
450 mL Shields and Sang M3 insect medium (Sigma #S3652)
(pour off 50 mL into Falcon to save as serum-free)
50 mL Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
5 mL 1:100 Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)
20 µg/mL Insulin (Sigma #I6634)
Sterile filter (0.2 µm) Store at 4°C - do not freeze

C. Clone8 (CL8) cells:

Complete M3 Medium
Shields and Sang M3 insect medium (Sigma #S3652)
2% Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
2.5% Fly extract (see below)
0.125 IU/mL (0.5mg/mL) Insulin (Sigma #I6634)
1x Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)
Sterile filter (0.2 µm)

D. Selective Media

Hygromycin media (to 200 ng/µL; a 1:2300 dilution)
450 mL Schneider’s medium (Gibco #11720-034)
(pour off 50 mL into Falcon to save as serum-free)
50 mL Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
5 mL 1:100 Penicillin-Streptomycin (Gibco #15070-063)(aliquoted in -20)
222.2 µL of Hygromycin B (Calbiochem 40051)
Sterile filter (0.2 µm). Store at 4ºC - do not freeze.
Cloudiness may appear after a few weeks. If cloudiness appear, discard the media and do not use on cells.

Puromycin media (to 5 µg/mL; a 1:2000 dilution)
450 mL Schneider’s medium (Gibco #11720-034)
(pour off 50 mL into Falcon to save as serum-free)
50 mL Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
5 mL 1:100 Penicillin-Streptomycin (Gibco #15070-063)(aliquoted in -20)
250 µL of Puromycin (Calbiochem 540411)
Sterile filter (0.2 µm). Store at 4ºC - do not freeze.
Cloudiness may appear after a few weeks. If cloudiness appear, discard the media and do not use on cells.

E. Heat Inactivating Fetal Bovine Serum
  1. Thaw the Fetal Bovine Serum on a shaker at 2-8°C (overnight)
  2. Pre-heat a waterbath to a temperature of 56°C. (Make sure the water covers all of the serum in the bottle)
  3. Heat the serum for 30 minutes in the 56°C bath.
  4. Allow serum to cool to room temperature before adding to cells.

 

2. INSULIN STOCK (100X)

  1. Dissolve 10 mg (25IU) in 0.5 mL of 0.01N HCL.
  2. Heat in 37°C to dissolve for few minutes.
  3. Add 19.5 mL of M3 Media to bring volume up to 20 mL.
  4. Filter Sterilize with a 0.22 µm filter.
  5. Aliquot and store stock at -20°C. Working stock may be kept at 4°C for 4-5 weeks.

 

3. FLY EXTRACT

  1. Start with a collection of 30-75 g of healthy OreR, CanS, or yw flies. These can be collected and frozen, dry at -80° C. Warm centrifuge to room temperature.
    1. To make 200 mL extract, use 30 g of frozen flies.
    2. To make 400 mL extract, use 60 g of frozen flies.
    3. To make 500 mL extract, use 75 g of frozen flies.
  2. Add weighed out flies to blender. Add appropriate amount of Shields and Sang M3 medium. Blend at medium to high speed until it looks as if all the flies have been lysed and the liquid is reddish from dispersed eye pigment.
  3. Transfer liquid with fly carcasses to 250 mL gasketted centrifuge bottles. Spin at 2600rpm in a GSA rotor for 20 minutes.
  4. Aspirate the fly bodies and oily layer off the top of the bottle.
  5. Transfer liquid to new 250 mL gasketted centrifuge bottles. Spin again at 3000rpm in a GSA rotor for 30 minutes
  6. Again aspirate off the oily layer on top. Transfer liquid to new centrifuge bottles.
  7. Heat Inactivate the fly extract by placing centrifuge bottles into a 60°C water bath for 30-40 minutes. You will see a precipitate form.
  8. Centrifuge the extract at 3200 rpm in a GSA rotor for 45 minutes. Precipitated material will form a grayish pellet.
  9. Filter the supernate. Filter through a 0.22µm filter in a tissue culture hood. For the first filter pass, place all of the glass pre-filters provided in the filter unit box on top of the 0.22µm filter. Hold them down with 2 pipettes as you pour the supernate into the filter unit. The fly extract tends to clog the filters, so you may need to use more than one filter unit. Once all the extract has been through at least one 0.22µm filter, aliquot into 12.5 mL aliquots and freeze in liquid nitrogen. Store at -20°C.

 

4. GROWTH CONDITIONS

Cells grow at 23-25°C and 33% RH
Do not need CO2
Split every 3-4 days to maintain
Density sensitive - die if too dense or too dilute

 

5. MAINTENANCE

Split one flask of cells (most recent date) into a new T-75 flasks (VWR# BD353136) when culture is confluent. Keep other flask as a backup. Monitor growth status by microscopy before splitting and decide whether to adjust recommended dilution factor accordingly.

A. Semi-adherent cell lines - will stick to new flask but come loose over time

S2*

3-4 days

1:3 - 1:4

S2c

3-4 days

1:3 - 1:4

Kc(167)

3-4 days

1:3 - 1:4

l(2)mbn

3-4 days

1:3 - 1:4

 

B. Adherent cell lines

DL1

3-4 days

1:8 (1:5 - 1:10)

DL2

3-4 days

1:8 (1:5 - 1:10)

S3

3-4 days

1:3

S2R+ and derivatives

3-4 days

1:8 - 1:10

CL8

4-5 days

1:5-1:10

BG2

4-5 days

1:3-1:4

BG6

4-5 days

1:3-1:4

 

C. Passing cells in flasks

Passing SL2, S2C, and S2* cells

  1. Detach cells from the flask either by banging or scraping
  2. Pipet cells up and down about 10 times to resuspend cells and separate clumps
  3. Aliquot cells into new flasks accordingly

Passing DL2, DL1, S2R+ (and derivatives), Kc167, Clone 8, and S3 cells

Protocol 1:

  1. Remove all medium
  2. Scrape cells with scraper
  3. Resuspend cells with 10mL of fresh medium, pipetting up and down about 10 times
  4. Aliquot cells into new flasks accordingly

Protocol 2:

  1. Remove medium
  2. Wash in PBS to remove any serum
  3. Add 5 ml trypsin; let sit 5-10'
  4. Bang cells off flask.
  5. Add 5 ml serum medium to inactivate trypsin and wash remaining cells off bottom of flask.
  6. Spin down cells at 1200-1400 rpm for 5 min.
  7. Resuspend cells in serum medium.
  8. Aliquot cells into new flasks accordingly

Protocol 3:

  1. Detach cells from the flask either by banging or scraping
  2. Pipet cells up and down about 10 times to resuspend cells and separate clumps
  3. Aliquot cells into new flasks accordingly

Protocol 4:

  1. Resuspend cells by thoroughly pipetting media in the flask onto the sheet of cells to dislodge them from the bottom until the sheet of cells is no longer observable and the media is cloudy
  2. Aliquot cells into new flasks accordingly

Passing BG2 cells

  1. Remove medium (save and filter through syringe filter for use as "conditioned medium").
  2. Wash in PBS to remove any serum
  3. Add 5 ml trypsin; let sit 5-10'
  4. Bang cells off flask.
  5. Add 5 ml serum medium to inactivate trypsin and wash remaining cells off bottom of flask.
  6. Spin down cells at 1200-1400 rpm for 5 min.
  7. Resuspend cells in conditioned medium.
  8. Aliquot cells into new flasks accordingly
  9. Can also use Accutase instead of trypsin.

D. Passing cells in wells - do not bang! Cells will spread to other wells within the plate and leak.

Protocol 1:

  1. Resuspend cells by thoroughly pipetting media in the well onto the sheet of cells to dislodge them from the bottom until the sheet of cells is no longer observable and the media is cloudy
  2. Aliquot cells into new wells accordingly

Protocol 2:

  1. Detach cells from the well by scraping with a scraper
  2. Pipet cells up and down about 10 times to resuspend cells and separate clumps
  3. Aliquot cells into new wells accordingly

E. Expanding S2R+ cells and derivatives

  1. Observe cells to determine confluency. If 100% confluent, cells are ready to be expanded.
  2. Resuspend cells in media and add full volume to the appropriate amount of media in the larger culture container.
  3. Expand cells in the following order: 96 well, 48 well, 24 well, 12 well, 6 well, T25 flask, T75 flask.
  4. Continue expanding until cells are in the desired culture container. Cells grow quickly when they are expanded and will likely need to be passed or expanded the following day

 

6. FREEZING

  1. Prepare numerous flasks of the cell line and grow to 70-80% confluency. Each cryovial will contain 1x107 cells.
  2. Label appropriate amount of 1.5 mL cryovials.
  3. Resuspend cells and combine in a conical tube.
  4. Count cells on the hemacytometer to determine concentration. Multiply by the total amount of mLs in the conical to determine the total amount of cells. This number divided by 1x107 is the total amount of cryovials that can be prepared.
  5. Spin down the conical at 1,200 RPM for 10 minutes.
  6. Aspirate off supernatant and resuspend at approximately 1x107 cells/mL in appropriate amount of freezing medium: FBS + 10% DMSO, filtered.
    1. Example: If you are freezing down 4 cryovials, you have ~4x107 cells. After spinning, add 3,600 µL of FBS and 400 µL DMSO.
  7. Aliquot 1 mL into each cryovial
  8. Wrap cryovials in paper towel, put in a styrofoam box, and place at -80ºC.
  9. After a few days, transfer cryovials to liquid nitrogen for long term storage.
  10. It is recommended to thaw a cryovial to ensure the freezing process was successful and that the cells grow.

 

7. THAWING

  1. Prepare T25 flask with 5 mL room temperature medium.
  2. Thaw cells quickly in room temperature water bath.
  3. Just before cells are completely thawed, decontaminate the outside of the cryovial with 70% EtOH.
  4. Transfer the cells to the T25 flask with 5 mL medium.
  5. Observe cells after a few hours. If a majority of cells adhered to the bottom, aspirate off the medium, wash cells gently with fresh medium without resuspending, aspirate off, and add 5 mL of fresh medium.
  6. Watch daily. Initially, cells may need to be split at irregular intervals.

 

8. COUNTING

  1. Clean the hemocytometer and cover slip with a KimWipe sprayed with 70% EtOH. Place the cover slip on top of the hemocytometer
  2. Resuspend cells in media and add 10 µL of suspended cells to the hemocytometer through the ridge below the cover slip to allow the cells to spread evenly across the surface.
  3. Using a microscope, locate the main hemocytometer grid and count the number of cells in the grid. The grid volumes represents 1/104 mL, so the concentration of cells in the original sample is NumberCellsCounted x 104 cells/mL.