DROSOPHILA TISSUE CULTURE

Don Rio

DROSOPHILA TISSUE CULTURE
Maintenance

We mainly use the Schneider line 2 cell line ;and the Kc cell line.; Both are diploid and originally derived from embryos. Schneider L-2 grow in M3 medium (see recipe) supplemented with 10% heat inactivated (56° C for 30 min) fetal calf serum and antibiotics (penicillin and streptomycin). Kc cells grow in D-22 medium with antibiotics (penicillin and streptomycin) but don't require serum. Both lines grow at 25°C without CO2 and are maintained in 25 cm² T-flasks (Corning). Routinely cells are grown at 1-5x10⁶ cells/ml and are split into fresh medium at a 1 to 4 or 5 dilution every 3 days. The cells don't attach well to a solid substrate and so are resuspended by pipeting for subculturing. No trypsin is required.

Freezing and thawing
1) Grow cells to 3-5x10⁶ cells/ml (log phase) in a 30-50 ml of medium in a 150 cm² T-flask.
2) Resuspend cells by pipeting (sterile) and transfer to 50 ml sterile polypropylene tube. Spin in IEC table top centrifuge (at setting 5) for 1-2 min.
3) Remove medium by aspiration and resuspend in 1.5 ml freezing medium. Use either M3 (for L-2) or D-22 (for Kc) and 10% DMSO (Schwartz-Mann, spectropure) and 20% H FCS.
4) Aliquot 0.5 ml cells into 2 ml nunc vials or provials. Label and transfer to freezer box with foam inside (to allow slow cooling).
5) Transfer to -70°C freezer overnight (or longer).
6) Transfer to liquid nitrogen tank for permanent storage.

To thaw:
1) Remove vial from liquid nitrogen and warm in water bath at 25°C (or room temperature).
2) As soon as the contents are thawed, transfer to 25 cm² T-flask with 5-10 ml medium.
3) Allow cells to attach (3 or more hrs) and replace medium with a fresh aliquot.
4) Incubate at 25°C for 3-5 days.
5) The cells may take a while to fully recover.
DROSOPHILA TISSUE CULTURE CELL TRANSFECTION

A. Solutions

1. 0.25 M CaCl2 :for 100 ml:
3.68g CaCl2 2 H2O
ddH2O to 100 ml

Sterile filter, aliquot into 15 ml polypropylene tubes. Store at -20°C.

2. 2XHEBS
for 1 L:
NaCl 16g
KCl 0.7g
Na2HPO4 0.4g
dextrose 2g
Hepes (free acid) 10g

pH to 7.1 with NaOH
ddH2O to 1 L
Sterile filter, aliquot in 50 ml tubes
Store at -20°C.

NOTE: When thawing for a transfection, re-pH to 7.1 and re-sterile filter before use.

B. Procedure
1) Seed 5 ml medium in 60 mm dish (Falcon) with 0.2 - 0.3 ml of cell culture (5-8x10⁶ cells/ml; e.g. 2-3 day old culture).
2) Incubate at 25°C at least 6 h or overnight before transfection.
3) Mix 10 µg plasmid DNA with 0.4 ml 0.25 M CaCl2 and add to 0.4 ml 2X HEBES pH 7.1 dropwise with swirling in 17x100 mm polycarbonate tubes (Falcon).
4) Incubate at room temperature for 20-30', the solution should become lightly cloudy.
5) Add 0.8 ml ppt/60 mm dish, swirl and incubate at 25°C.
6) For selection: (e.g. for transformed cell lines) after 24 h: split cells 1 Æ 4 into new medium. After another 24 h add selective drug. G-418: (for pcopneo and derivatives) at 1 mg/ml; Hygromycin (pcophyg and derivatives) at 200 µg/ml.
7) Split cells every 7-10 days into selective medium.
8) Grow cell lines as mixed cultures in selective medium.
ECHALIER'S D-22 MEDIUM FOR Kc CELLS (HIGH PO4) ;- 10L - 2X.D22

1) Dissolve in less than 500 ml
L-glutamic acid 79.4g
glycine 40.4g
Adjust to pH 7.0 with KOH pellets (about 37.5 gm; not much more, if any!)

2) Dissolve in less than 500 ml
L-glutamic acid 138.18g
glycine 70.32g
Adjust to pH 7.0 with NaOH pellets.

3) Dissolve in 250 ml or less
MgCl2 6 H2O 20g
MgSO4 (anhydrous) 36g

4) Dissolve in ~150 ml
Na H2 PO4 H2O 8.32g

5) Dissolve in less than 500 ml
L-malic acid 13.4g
succinic acid 1.2g
sodium acetate 3 H2O 0.5g
glucose 40g
phenol red (sodium salt) 0.2g

6) Dissolve in 1 L
CaCl2 17.8g

7) Vitamin de Grace - 500X 40 ml

8) Dissolve in 1 L
yeast extract 30g

9) Dissolve in 4 L
Heat to dissolve. Cool and add slowly to media.
Lactalbumin hydrolysate 300g

Mix in this order:

1) Add CaCl2 (6) to MgCl2 and MgSO4 (3) slowly with stirring on stirrer.

2) Add K-glutamic/glycine (1)
Na-glutamic/glycine (2) and Na H2 PO4 H2O (4)
malic acid/succinic acid/glucose/NaOAC (5)
yeast extract (8) and lactalbumin hydrolysate (9)
500X graces vitamins (7) then add PEN/STREP

for 10 L 2 X - add 1.0g streptomycin sulfate
0.63 g penicillin G 1585u/mg

3) Adjust to pH 6.7 with KOH
4) Adjust volume to 10 L
5) Filter sterilize and dispense into sterile 1L bottles.
6) Store at 4°C.

500X GRACE'S VITAMINS FOR ECHALIER'S D22 MEDIUM

Vitamin mg/L

thiamine hydrochloride riboflavin 10
calcium pantothenate 10
pyridoxine hydrochloride 10
p-aminobenzoic acid 10
folic acid 10
niacin 10
isoinositol (myo-inositol) 10
biotin 5
cholin chloride 100
M3 MEDIUM - FOR SCHNEIDER LINE-2 CELLS:; 10L (1X)

gg
a-alanine 15 oxaloacetic acid 2.5
b-alanine 2.5 phenylalanine 2.5
arginine-HCl 6..04 proline 4
asparagine 3 serine 3.5
aspartic acid 3 threonine 5
cysteine-HCl 2 tyrosine 2.5
glutamine 6 tryptophan 1
K glutamate 71.5 valine 4
Na glutamate 65.3 glucose 100
glycine 5 bactopeptone 25
histidine 5.5 yeastolate 20
isoleucine 2.5 CaCl2 7.6
leucine 4 MgSO4 21.5
lysine-HCl 8.5 Na H2PO4 H2O 7.8
methionine 2.5 BIS-Tris 10.5

Dissolve in 9 L dd H2O
Add 1 mg choline and 10 mg KHCO3
Adjust pH to 6.8 with NaOH, adjust volume to 10 L
Filter and store at 4°C

For complete medium add heat inactivated fetal calf serum and pen/strep.

To make radiolabeling medium (for 35S-met): omit methionine, yeastolate and bactopeptone.