Biology Reference
In-Depth Information
FBS-1.5% penicillin-streptomycin containing medium. Seal the plates with
parafilm and Saranwrap, and grow cells at 25
C for 3 days.
4.
Dilute oleate/BSA stock (7.5 mM) to 2 mM in 10% FBS-1% penicillin-
streptomycin containing medium. Add 100
l/well (1 mM oleate final
concentration) to the plates and incubate at 25
C for 1 more day.
5.
Prepare Con-A-coated glass-bottom plates: 1 day before use, coat 96-well glass-
bottom plates with 40
m
g/ml Con-A solution, incubate at 30
C
m
l/well of 50
m
until absolutely dry.
6.
Resuspend cells thoroughly by pipetting up and down at least 20-30 times
(takes at least 2 h for 10 plates), try to avoid foaming. Transfer 30
m
l(
1/6) of
total cells to Con-A-coated glass-bottom plates, add 20
m
l medium, and incubate
at room temperature for at least 120 min.
7.
Add 50
m
l 8% formaldehyde/2
PBS to fix at room temperature for 1 h,
wash with 1
PBS twice, stain with 2
m
g/ml BODIPY493/503 and 0.3
m
M
DAPI in 1
PBS for 1 h, wash with 1
PBS twice, add 100
m
l/well 50%
20
C.
glycerol/1
PBS. The plates can be imaged at this time or stored at
8.
Image the plates on automated microscopes using a 40
0.95 numerical
aperture PlanApo dry objective lens (Nikon) for both BODIPY493/503 and
DAPI signals. Six image fields were taken per well to image 400-600 cells per
RNAi condition.
9.
Score LD number, size, and cellular distribution visually by two independent
observers who are blinded to gene identities for the 95,000 images, and also
analyze the images by automated image analysis (
Guo et al., 2008
).
10.
Synthesize dsRNAs targeting different regions in the 847 genes identified from
the primary screen and repeat (1-9) to confirm the primary screen hits.
Notes
: We routinely use several control dsRNAs (
Pavarotti
for cell morphology,
mid-
way
or
Cct1
for LDmorphology) to batch test the cell culture medium and FBS before
performing any RNAi experiments. For unknown reasons, the S2 cells do not grow
well in some batches of the medium or FBS, and thus the RNAi efficiency can be low
under those circumstances. More detailed discussion about S2 cell RNAi experiments
in general can be found in previous reviews (
Bettencourt-Dias & Goshima, 2009;
Goshima, 2010
). For our screen, we pre-reserved adequate amounts of batch-tested
medium and FBS to ensure the whole screen was performed with the same culture
medium. Under optimal culture conditions, 90% confluent S2 cells from one
225 cm
2
tissue-culture flask are enough to seed ten 96-well screening plates. We
propagated an adequate amount of cells and stacked the screen procedure so the entire
screen was seeded within 2 weeks to ensure the cells were from similar passages.
4.1.5
Screening results and discussion
In our primary screen, 15,683
Drosophila
genes were knocked down individually in
S2 cells to screen for novel regulators for LD biology. We combined the visual screen
by two independent observers and computational analysis to score changes in LD
