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  • Bruno Vellutini 21:08 on 2013/08/10 Permalink
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    4D Membranipora 25 µM U0126 10°C 

    Folder: BCV_Mm_2013_08_10

    4 cell stage from 25 µM treatment at 10 °C. Recording is not so good and embryo is disrupted super early with 8 cells.

     
  • Bruno Vellutini 20:12 on 2013/08/10 Permalink
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    First inhibitor experiment U0126 

    With the huge spawning I decided to set the first MAPK inhibitor experiment. I setup 6-well plate with different concentrations of U0126. Since we only had one aliquot on the freezer (who knows how old; from 2009) I got one 1 mg glass from S4 and made a fresh dilution; it is a Promega kit.

    CONTROLno DMSO

    5 mL FSW

    CONTROLDMSO 1:400

    12.5 µL in 5 mL FSW

    U0126 1 µM1:10000

    0.5 µL in 5 mL FSW

    U0126 10 µM1:1000

    5 µL in 5 mL FSW

    U0126 25 µM1:400

    12.5 µL  in 5 mL FSW

    I made two identical setups with the inhibitor added 3h post-activation and kept at 10 °C:

    1. Embryos picked without much selection (activated: 16h; inhibitor: 19h)
    2. Only healthy 2 cell embryos were picked (activated: 17h; inhibitor: 20h)

    Also set a 4D recording with a 4 cell stage from 25 µM treatment.

    11/08/13

    20h later embryos in the controls look healthy at blastula/gastrula stage, no visual differences from the controls, 1 µM and 10 µM. 25 µM samples were disrupted with development halted during cleavage stages. Fixed about half of 22h samples for immunostaining.

    4D was disrupted at 8 cell stage, but recording not very nice.

    12/08/13

    44h later embryos had a much clearer differences. Controls had a large number of swimming late gastrulae as well as the 1 µM concentration of U0126 (although not quantified yet). 10 µM had only round embryos in the fine picked plate, as if they were stuck in the blastula/gastrula stage and close to none swimming gastrula. 25 µM continued disrupted.

    Fixed that rest of the samples.

     
  • Bruno Vellutini 18:52 on 2013/08/10 Permalink
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    Membranipora spawning 

    I tried spawning colonies from the bowls in the cold room and they were not looking good. Zooids were full of eggs, but they were not active and many looked dead. After many hours outside they didn’t come out. So I scratched some and they seemed to develop.

    However, I took a small piece of kelp from the leaves that are in the S4 flowing tanks and 5 minutes after they were spawning like crazy! Without even keeping in the dark. This piece rendered 4 bowls of embryos, literally millions of embryos, much more than last time.

    So, keeping them in the flowing tank is key!

     
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