4D with U0126 Membranipora
15 °C, 15 µM
BCV_Mm_2014_09_23 (old Mmem1_23_09_2014)
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Bruno Vellutini
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Bruno Vellutini
4D with U0126 Membranipora
BCV_Mm_2014_09_20 (old Mmem1_20_09_2014)
10 µM, 15 °C
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Bruno Vellutini
Membranipora U0126 inhibitor time windows
I set the U0126 inhibitor 10 µM with Membranipora at 15 °C. I am washing every 2 hours after 1h30 hours after activation as shown below (2x, for 24h and 48h fixation).
- Activated: 1100
- Inhibited: 1230
- 1st wash: 1430
- 2nd wash: 1630
- 3rd wash: 1830
DMSO 3.5 hpa 5.5 hpa 7.5 hpa 20/09/14
Fixed 24h.
21/09/14
Fix 48h.
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Bruno Vellutini
Membranipora 4D U0126 10 µM
Started a recording BCV_Mm_2014_09_18 (old Mmem1_18_09_2014) with Membranipora in 10 µM U0126 MEK inhibitor at 15°C. Embryo is looking quite good and stable.
19/09/14
Embryo is still lokking good and healthy (?). Letting it on… Stopped and saved the embryo. It moved out of the field of view around 1700.
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Bruno Vellutini
Membranipora 4D U0126 10 µM
Started a recording BCV_Mm_2014_09_17 (old Mmem1_17_09_2014) with Membranipora in 10 µM U0126 MEK inhibitor at 15°C. Embryo is looking quite good and stable.
19/09/14
Well, all the embryos were dead. Cells exploded and all. I think this was due to the high temperature of the room which made the slide temperature not be 15 °C as the ring should be, but more, killing the embryos.
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Bruno Vellutini
Membranipora and U0126 inhibitor fine picked
Set up another U0126 inhibitor experiment. This time I’m using a fine picked and a coarse picked set of embryos. The setup is the same, but I picked only healthy 2-4 cell stage for the fine picked and a less strict sampling for the coarse picked (but still trying to fetch only good embryos.
FSW DMSO 5 µM 7.5 µM 1 µM 2.5 µM 10 µM 12.5 µM Embryos were activated at 11h, picked from 12:30-13:30 and incubated at 13:30 (fine) and 14:00 (coarse) in the facility incubator at 15 °C!
19/06/14
Fixed after 24h.
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Bruno Vellutini
Another Membranipora U0126
Another Membranipora spawning and I set up a new U0126 inhibitor experiment. I made 2 replicates again of the following setup:
FSW DMSO 5 µM 7.5 µM 1 µM 2.5 µM 10 µM 12.5 µM Embryos were activated at 12h and incubated at 15h in the facility incubator at 15 °C!
I’ll fix the embryos on Tuesday after 24h.
17/06/14
Fixed after 24h.
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Bruno Vellutini
Membranipora U0126 experiment with different concentrations
With the nice Saturday spawning I managed to setup a new U0126 inhibitor experiment. I made 2 replicates of the following setup:
FSW DMSO 5 µM 7.5 µM 1 µM 2.5 µM 10 µM 12.5 µM Embryos were activated at 14h and incubated at 17h in the facility incubator at 15 °C!
I’ll fix the embryos on Monday after 48h.
16/06/14
Replicate one everything was dead except for FSW control, very weird. The second replicate seemed fine, although there should be a lot of noise. I’ll repeat the same today and fix 24h after.
I’ll fix with 48h.
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Bruno Vellutini
First inhibitor experiment with L. viridis
Auxane and me set an inhibitor experiment with DMH1 and U0126 with early cleaving embryos from Lineus viridis. We dissociated the packets from the gelatinous layer and incubated with the following treatments.
DMH1 1µM DMH1 5µM U0126 1µM U0126 5µM DMH1 10µM DMSO (1%) U0126 10µM FSW 02/04/14
No apparent difference between the experimental samples. Similar amounts of normal/weird/non-cleaving embryos.
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Bruno Vellutini
Membranipora MAPK U0126 quantification
Before staining for the phenotype description I counted the number of normal embryos in the fine picked samples. Normal means late gastrula with apical lobe and elongated body axis.
Seawater DMSO 1µM 10µM 25µM Normal late gastrula 31 31 33 0 0 Weird 6 11 10 40 23 Total 37 42 43 40 23