Research Update: Sofie's Lab Book Journey from October 2018 to December 2019
Sofie's research progress includes training on C. elegans and drosophila, meetings with various groups, preparing for conferences, and application for funding. She is focused on stem cell research, premature aging studies, and biomarker analysis in CSF.
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Sofies Monday update /lab book From October 2018 to December 2019
Last week: 10/01 10/12 General training/introduction has been completed. Training on C.elegans: Distinction of L4 stage (test) Learnt to pick up worms Learnt nomenclature Learnt on maintance of worms WB with Yahyah of Tau and Abeta of worm extracts Premature aging Werner syndrome in drosophila: Read papers (also for WS review) Training on flies with Roji (Radium Hospitalet) Flipped flies Monday and Thursday Learned nomenclature I contacted Costas and Pete about metabolics in the flies send by Nica in August no reply yet iPCS-induced neurons: Skype with Mahdi s friend on possibility of creating WS iPSC lines Meetings: Section meeting with Tor Erik s group and section at Radium Hospitalet (Monday) Patty Lee (Monday) Group meeting: presented PhD work + techniques (Tuesday) Nansen Neuroscience lecture (Wednesday) + wrote report for web-page Skype with Bohr (Wednesday) EpiGen group meeting (Friday) I signed up for the Keystone Conference (J2) and booked Hotel Send doodle for Autophagy meeting This week: 10/15 10/19 Training on C.elegans: Distinction of L4 stage Keep on training on learn to pick up worms every day Premature aging Werner syndrome in drosophila: Read papers (finish my part of the WS review) Prepare ppt for October 23rd (autophagy supergroup meeting) Continue flipping/maintaining flies (Monday and Thursday) Read and prepare protocols and optimization of stem cell measure contact Martin if necessary Others: Start application for hosting a meeting funding (internal deadline for Evandro is 8th of November) Make Mobility/Conference application (ReNEW + potentially others) Meetings Skype with Bohr: will be on C. elegans work in Baltimore Order food for Autophagy meeting
Last week: 10/15 10/19 Training on C.elegans: Distinction of L4 stage (test) Maintance of worms, pick up every second day in the lab Preparation of M9 buffer with Tanima Preparation of fresh plates with Guofeng Premature aging Werner syndrome in drosophila: Finished first combined draft of review + figures Maintain and expand flies WT and WRNexo flies (Radium Hospitalet) Start expansion of additional lines: LacZ RNAi, PINK1 RNAi, BNIP3 RNAi lines (I have written Martin to ask if the BNIP3 is the same as BNIP3L it is). Polishing stem cell proliferation protocol + contacted Martin Still no reply from Costas and Pete about metabolics in the flies send by Nica in August CSF Biomarkers ELISA kits for PINK1 examined + overview made (attached) Checked litterature for ANY studies on PINK1 proteomic studies in CSF cannot find any Funding: Applied for ReNEW Started my application for hosting a meeting (internal deadline Evandro is 8th of November) This week: 10/22 10/26 Training on C.elegans: Distinction of L4 stage Maintain worms Read about basics of C elegans from Evandro including nomenclature. Premature aging Werner syndrome in drosophila: Correct WRN review Continue flipping/maintaining flies (Monday and Thursday) Prepare al buffers for optimization of stem cell proliferation next week (then I think I will have enough flies to make a trial) Get training with confocal. Tanima? She did not have time. CSF Biomarkers Order kits Make litterature review with Guofeng Others: Finialize application for hosting a meeting funding (internal deadline for Evandro is 8th of November) Orderings: The 25th of October 2018: ordered 3 ELISA kits (for PINK1, ULK1, Parkin) via Ellen W., UiO account.
Labbok notes: week 21-26/10 2018 Training on C.elegans: Distinction of L4 stage Maintain worms Read about basics of C elegans from Evandro including nomenclature. How to synchronize and get many worms at same state: Bleach Filter Grow many plates, pick worms at same stage Transfer adults (1 or 2), leave them on plate to lay eggs for 2 hours, remove them then all the eggs will hatch approx. Same time. Premature aging Werner syndrome in drosophila: Correct WRN review + prepare stem cell figure Continue flipping/maintaining flies (Monday and Thursday) Flies: Thursday the 18th of October I started the expansion on LacZRNAi, PINK1RNAi, BNIP3RNAi. They are growing fine and laying eggs. Prepare al buffers for optimization of stem cell proliferation next week (then I think I will have enough flies to make a trial) I will use NICA s stocks, and prepare fresh BSA and glutamic acid just before use next week. Practive the dissection of guts of DaGs flies and anestezise with CO2 (25/10/2018). CSF Biomarkers Order kits 2018-10-25 from Ellen Westergard (UiO) Kits ordered: ULK1 BioNordic, PINK1 and Parkin ELISA MyBioSource, 96 well plates Make litterature review with Guofeng I have started with PINK1. Others: Finialize application for hosting a meeting funding (internal deadline for Evandro is 8th of November) Skype with Albert (2018-10-26) on WS, iPSCs, ESCs, MSCs he will provide us with all three types of cell lines. Gut, cleaned from ovaries, crop etc. Gut from female fly Gut and ovaries from female fly Fly body
Stem cell protocol In vivo stem cell proliferation assay (By Nica Caponio, and Sofie Lautrup, according to Martin suggestions) Plate bacteria on LB plate to grow colonies, ON 37C. Inoculate, grow ECC15 bacteria in LB (without any antibiotic) aerobically at 37 degrees overnight. How to set up flies in food with bacteria (feed ecc15 it's in purely liquid food): Rip a Kim wipe in 1/8ths in the vial Put Whatman filter on the top (https://www.sigmaaldrich.com/catalog/product/aldrich/wha1003323?lang=en®ion=US) Pellet 15 ml of ECC15 culture at 1000 rpm in a big swinging arm centrifuge Resuspend the pellet in 500 l of sucrose 5% Add 500 ul sucrose solution +/-ECC15 (use only sucrose for control) Add flies (15 guts/condition are enough) Incubate for 24h The day after dissect guts in 1X phosphate-buffered saline (PBS) Fix for 45 min at room temperature (100 mM glutamic acid, 25 mM KCl, 20 mM MgSO4, 4 mM sodium phosphate, 1 mM MgCl2, and 4% formaldehyde) Wash for 1 h at 4 C (1x PBS, 0.5% bovine serum albumin and 0.1% Triton X-100) Incubate with rabbit anti-phospho-Histone H3 Ser 10 overnight at 4 C in wash buffer. Wash 3x 10 min Incubate with secondary antibody ( -rabbit) (Yahyah uses 1:500) at room temperature for 2 h (protected from light) Wash 3x 10 min. Labbok notes: week 21-26/10 2018 Preparation of buffers LB without antibiotics: the LB medium we get from the kitchen on the fourth floor. Use 15 mL to one scrabe bacteria from freezer 5% sucrose: Nica has prepared some in end of June, it is at Radium and it looks OK. 1xPBS: in Fang lab at Ahus we have 10x PBS, dilute to 1x and bring to Radium. Fixation buffer: Wash buffer (prepare fresh, keep on ice/4C until use) Antibodies: Primary antibody: rabbit anti-phospho-Histone H3 Ser 10 -From where, catalog number, lot number: -Nica used 1:8000. I think it is too much diluted, so will try with 1:1000, since this is what is used in most papers (see below). Try with 1:4000 and 1:1000. Secondary: -rabbit fluorescent which tag/color? -From where, catalog number, lot number: Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 . -1:500 (Yahyah), try different dilutions of the secondary 1:500 and 1:1000. Mounting medium? Should be in -20C freezer? References for Stem cell in midgut of drosophila: (Jin, Ha et al. 2015): they use 1:1000 pH3, 1:1000 secondary antibody. They only use the midgut, not the complete instestine. They normalize to DAPI. (Patel, Dutta et al. 2015): they use 1:1000 pH3, also stain senescence by bGal antibody. They use only the midgut complete intestine. They also stain DAPI. They use same wash buffer as above, but block in wash buffer with 2% normal goat serum. Additional explanations from Nica 2018-10-18 References Jin, Y., N. Ha, M. Fores, J. Xiang, C. Glasser, J. Maldera, G. Jimenez and B. A. Edgar (2015). "EGFR/Ras Signaling ControlsDrosophila IntestinalStem Cell Proliferationvia Capicua-RegulatedGenes." PLoSGenet 11(12): e1005634. Patel, P. H., D. Duttaand B. A. Edgar (2015). "Niche appropriationby Drosophilaintestinalstem cell tumours." Nat Cell Biol 1182-1192.
Monday update week 21-26/10 2018 Training on C.elegans: Distinction of L4 stage, maintain worms Read about basics of C elegans from Evandro including nomenclature. Premature aging Werner syndrome in drosophila: Corrected WRN review + prepared stem cell figure Continue flipping/maintaining flies (Monday and Thursday) Practice the dissection of guts of DaGs flies and anestezise with CO2 (see below images) CSF Biomarkers Order kits 2018-10-25 from Ellen Westergard (UiO) Kits ordered: ULK1 BioNordic, PINK1 and Parkin ELISA MyBioSource, 96 well plates Make litterature review with Guofeng I have started with PINK1. Others: Finialize application for hosting a meeting funding (internal deadline for Evandro is 8th of November) Skype with Albert (2018-10-26) on WS, iPSCs, ESCs, MSCs he will provide us with all three types of cell lines. Week 29/10-2/11 2018 Training on C.elegans: Distinction of L4 stage, maintain worms, study the basics of C. elegans Premature aging Werner syndrome in drosophila: Figure legends for review figures + send to coauthors Continue flipping/maintaining flies (Monday, Wednesday, friday) Start crossing the flies Optimization experiment with stem cell proliferation measures Confocal microscopy training with Tanima In human cells Arrange shipping of WS cells with Albert Gut, cleaned from ovaries, crop etc. CSF Biomarkers Make litterature review with Guofeng I have started with PINK1. Gut from female fly Gut and ovaries from female fly Fly body
Lab book Week 29/10-2/11 2018 Training on C.elegans: Distinction of L4 stage, maintain worms, study the basics of C. elegans Tuesday and Friday Premature aging Werner syndrome in drosophila: Figure legends for review figures + send to coauthors (Monday sent to Albert) Continue flipping/maintaining flies (Monday, Wednesday, friday) Start crossing the flies (Monday 29th WRNexoRNAi x DaGS) Optimization experiment with stem cell proliferation measures Sunday 28th: plate bacteria, grow ON Monday 29th: pick one colony, grow ON in LB buljong 37C with shaking. Flies: Flip and put the DaGS flies in the big bottles. Confocal microscopy training with Tanima Tuesday: take 15 mL bacteria to Radium, spin down 4000xG RT to pellet, resuspend in 500 uL 5% sucrose, add to vial with fiter paper and filter (vehicle with sucrose solution alone) Add 12 female DaGS to +ECC15, 6 to Veh, leave ON (see images below). Flip LacZ, PINK1, BNIP3 (did not have time Monday) Prepare buffers: wash buffer, fixation buffer, 1xPBS, 10% BSA, 500 mM Glutamic acid necessary to adjust pH to 6 to dissolve (done with NaOH). See protocol for stem cell proliferation for details. Wednesday: dissect guts from flies treated with bacteria: 10 flies with bacteria, 3 flies without, followed stem cell proliferation protocol. Primary antibody: either 1:4000 or 1:1000 ON 4C. Also, added one virgin DaGS to the crossing, since 3 females died already. Thursday: continue stem cell proliferation protocol. Secondary antibody (4C storage) 1:1000 or 1:500. used ProLong Gold Antifade reagent with DAPI for mounting. Imaged with help from tanima on the LSM Zeiss Confocal Microscope. Images saved with the following names: 2018-11-01 1 4000. 2 1000 +E : means primary antibody dilution 1:4000, secondary 1:1000, flies infected with ECC15. 2018-11-01 1 4000. 2 1000 +E 1.7crop: the zoomed version of the same images as before. If it says minus E: non-infected flies. Conclusion: the antibody dilutions with 1:1000 primary and 1:1000 secondary was better than 1:4000. Though it looks like the tissue is not completely permeabilized or might have dried out. Next time: add a bit more triton to the wash buffer, and make sure that the tissues on the slides do not dried out before adding mounting media. Friday: flip flies (all vials). Started one additional crossing vial: 5 virgin DaGS + 1 male WRN. Added 2 extra virgins to already exiting crossing vial. Notes: only 1 WRNexoRNAi male without curly wings? Perhaps I need to start using both curly and non-curly males? Enough flies to start large vials with BNIP3, PINK1, LacZ next week? WRNexoRNAi in large bottles next week? Perhaps not enough flies. In human cells Arrange shipping of WS cells with Albert (wrote Albert Tuesday 30th) Wrote to the company that we are finding out the last details (Tuesday 30th) The full name of RU CSF Biomarkers Make litterature review with Guofeng I have started with PINK1. Guofeng is preparing setup and protocol for the kits.
Lab book Week 29/10-2/11 2018 Optimization experiment with stem cell proliferation measures Sunday 28th: plate bacteria, grow ON Monday 29th: pick one colony, grow ON in LB buljong 37C with shaking. Flies: Flip and put the DaGS flies in the big bottles. Confocal microscopy training with Tanima Tuesday: take 15 mL bacteria to Radium, spin down 4000xG RT to pellet, resuspend in 500 uL 5% sucrose, add to vial with fiter paper and filter (vehicle with sucrose solution alone) Add 12 female DaGS to +ECC15, 6 to Veh, leave ON (see images below). Prepare buffers: wash buffer, fixation buffer, 1xPBS, 10% BSA, 500 mM Glutamic acid necessary to adjust pH to 6 to dissolve (done with NaOH). See protocol for stem cell proliferation for details. Wednesday: dissect guts from flies treated with bacteria: 10 flies with bacteria, 3 flies without, followed stem cell proliferation protocol. Primary antibody: either 1:4000 or 1:1000 ON 4C. Thursday: continue stem cell proliferation protocol. Secondary antibody (4C storage) 1:1000 or 1:500. used ProLong Gold Antifade reagent with DAPI for mounting. Imaged with help from tanima on the LSM Zeiss Confocal Microscope. Images saved with the following names: 2018-11-01 1 4000. 2 1000 +E : means primary antibody dilution 1:4000, secondary 1:1000, flies infected with ECC15. 2018-11-01 1 4000. 2 1000 +E 1.7crop: the zoomed version of the same images as before. If it says minus E: non-infected flies. Conclusion: the antibody dilutions with 1:1000 primary and 1:1000 secondary was better than 1:4000. Though it looks like the tissue is not completely permeabilized or might have dried out. Next time: add a bit more triton to the wash buffer, and make sure that the tissues on the slides do not dried out before adding mounting media Images from optimization experiment: DAPI merged DAPI merged
Toms Schmauck Monday Update From 21 Sept. 2020 to 8th Feb. 2021 Intelliblog: EOS AND TITHONUS Eos and Tithonus. Tithonus was a mortal prince and Eos a Goddess, they were lovers. Eos asked Zeus to make Tithonus immortal. But forgot to ask for eternal youth. Tithonus lived and AGED forever, till he turned into a cicada.
Picture of the Week Knife swallowing. This X-ray shows a knife that became stuck in a 30- year-old woman's esophagus and stomach. The woman suffered from bulimia, and had inserted the knife into the back of her mouth in order to demonstrate that she had lost her gag reflex. But she laughed unexpectedly, and the knife fell into her body. Doctors were able to remove the knife in a procedure called an esophagogastroduodenoscopy.
LAST WEEK (22/02/2020 28/02/2021) WORK o Created more RNAi for seeding o Seeded plates with RNAi bacteria for BAG-1 experiments o Soon to conclude the double generation initial experiment o Did a REST memory experiment by myself, but the results seem off. I will do another one this week. o (spr-4 mutation, spr-4 overexpression, WT, spr3 and 4 double mutant) o Shu taught me how to use the fluorescent microscope for tau Venus photograph o Expanding BAG-2 worms for experimentation o Prepared more RNAi plates for next week o Doing an immunohistochemistry for REST o Bleached JKM strains o Sunybiotech contract o Crossing schematic o Helped Alice rehearse her presentation MEETINGS o Tuesday o Lab meeting o Presenting journal club extra paper o Thursday o NO-AGE NO-AD seminar READING o Parkin-dependent mitophagy protects macrophage against oxidative injury and inflammation during atherogenesis o Reviewing the paper o A protein factor essential for microtubule assembly o Seems to be one of the first (if not the first) papers on tau o The Role of Tau in Alzheimer s Disease and Related Disorder o Review on Tau
UPCOMING WEEK (08/03/2020 04/03/2021) WORK o Memory on BAG-1 RNAi if the worms reach D1 (likely) o Will do another REST memory experiment o (spr-4 mutation, spr-4 overexpression, WT, spr3 and 4 double mutant) o Freezing JMK strains o Bleaching the new strains (p62 strains, LC108, etc) o Understanding how JMK strains aggregation quantification works o Finish the review for the Aging paper MEETINGS o Monday o NO-AGE NO-AD seminar o Tuesday o Lab meeting
