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I'm looking information about some experimental aspects on Drosophila melanogaster, is it possible to know the physiological pH of this study model?
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It can be evaluated through the homogenates of flies and pH did not change with time after homogenization. Study flies can homogenize for 30 sec at 25 °C in 1 ml of air saturated distilled water with a motor-driven (1800 RPM) glass-teflon homogenizer (clearance 0.13-0.18 ram) and dilute to 10 ml for measuring pH by glass electrode.
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Hello,
I need to identify a protein that interact with an enhancer and maybe with a promoter (the protein allows the enhancer to activate the promoter)
Do you have an idea how I could identify this protein ? Thank you
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I have heard that dipping fly heads in 70% ethanol before placing in cold PBS for dissection can help strip the heads of wax and allow them to stay submerged in the PBS during dissection. I was wondering if doing this step can cause complications for protein extractions or even imaging the dissected brains using confocal microscopy? Thanks in advance for anyone's time in answering this.
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Hello ,,Nicholas
Maybe this PDF helpful for you..
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Hi everyone I was wondering if anyone had used the Jazz Mix Drosophila food from Fisher or WARDS instant Drosophila media from VWR in their research, and if you find it convenient and easy to use, and does the flies seem happy with it? If you have used it, do you continue to use it? Makes your life easier? Or if you don't like it why? Which one do you prefer if you have tried both?
Thanks for all the feedback I will really appreciate it :)
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Came across this while having breakfast with my younger daughter and thought I'd add a bit of history to Jazz mix. I formulated this some years ago for Grier Eubank's company (I don't recall its name), which was subsequently bought by Fisher. Our purpose was to get micronutrients into the food, which helps weak stocks. Of note, the name of my daughter is...Jazz.
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Hey everyone, 
I have a few quick questions.  I want to do subcellular fractionation on Drosophila S2 cells.  In your experience, does the Pierce Subcellular Protein Fractionation kit work on insect cells- the website says they are for mammalian cells (both suspension and adherent)?  Or do you have other kits or protocols you recommend? 
My second question is on what is a good control for something that would be in the nuclear fraction but would not bind to chromatin.  E.g. I've read that for the nuclear fraction, people often use Histone H3 as a control.  But I am also interested in separating my nuclear fraction into: chromatin-bound in the nucleus vs. not bound to chromatin in the nucleus.  What would be a good control for this?  
Thanks so much!!!  
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hi, it is from Sigma if I remember correctly.
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I am doing a project on trying to stain and image the neuromuscular junctions of drosophila leg muscles in great detail (with the possibility of imaging the boutons as well). So I have got two questions pertaining it:
1. I am going to fix the leg tissues in PFA but I have got contradicting protocols whereby some instruct to fix overnight at 4 degrees celsius while some simply instruct to fix at room temperature for 20-23 mins. Does this really make a difference or would doing the latter be sufficient? Also, I would like to know if fixing at a low temperature overnight might create any negative consequences on the results.
2. We have tried imaging the NMJs of adult drosophila legs before, whereby on anti-HRP was used as the only primary antibody. Several papers that I have read through have also mentioned anti-Dlg and anti-Brp as good candidate antibodies to visualize the boutons with greater precision. However, at the moment, we do not have anti-Dlg and anti-Brp so I am thinking of trying out another antibody to stain. Would anti-vGlut be a good choice of primary antibody to visualize the boutons?
Thank you in advance!
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Elizabeth Van Pelt-verkuil Oh I see! I am sorry I misunderstood that.. But thank you anyways!
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I am having trouble with the quantity of eggs I am collecting for microinjections as I only seem to get anywhere from 0 to 10 eggs every 30 minutes as opposed to hundreds. I make sure to have flies in the chamber that are about a week old (3 to 9 days), try to keep a large number of flies in the chamber, leave a large smearing of yeast on egg plates, change egg plates constantly before injections, and keep the flies at 25C, 70% humidity. I am currently using flies transgenically expressing Cas9 (from Bloomington) and wild-type flies that have been recently caught in the wild in the last few years. Any suggestions would be appreciated!
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Hello,
in adition to the yeast advices, for me used to work well to spread, on the surface of the agar medium, some drops of acetic acid at 10%. The smell makes them lay more eggs. Good luck.
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Kindly suggest me some tips or precautions to avoid fungal contamination in Drosophila culture tubes. In my case contamination persists even after flipping of flies, and contamination seriously affects growth of larvae.
Regards
Asif
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I used propionic acid, it works very well against fungal growth ... I recommend it for you
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I am planning to isolate stem cells from drosophila ovary. I will like to know what will be a suitable method to isolate the stem cells and what are the specific markers in order to detect the stem cells in drosophila ovary.
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Which stem cell population are you interested in -  the germline stem cells or the follicle stem cell population? 
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Hi All,
We found a population of infected adult Drosophila suzukii dying from E. muscae, we think. The flies originally were found on fig fruit and were motionless. Today, sporulation was observed. Is their a convenient method of plating and culturing this or other related entomopathogenic fungi. Might simple PDA media suffice?
Hope you can help.
All the best Blair    (blair.sampson@ars.usda.gov)
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I knock down a gene, the fly can survive in starved food, but next generation can't lay eggs.. 
that means, the flies can survive well and lay eggs in reach food. when transfer them in the starved food, all the flies can lay eggs, wait  the flies come out, transfer the fresh flies into a new starved food vial, they can't lay eggs. the gene effect the larvae? 
we have another gene, when knock down, in starved condition for couple hours, the ovary effected and can't lay eggs. 
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Are you working with genes influencing Insulin signaling? Insulin signaling is reduced upon starvation, resulting in lower fertility. Interestingly, according to your description, this effect is passed on to the offsprings, indicating epigenetic modifications. Have you checked if the methylation or acetylation status of the chromatin is affected?
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Hi,
I am looking for tips on how to rescue a line that is contaminated by  a fungus. This is only in one line out of about 40 that we have, and comes up in any cross from that line. We add nipagin M and propionic acid to our food (added after it cools after cooking), and we also add brewer's yeast to the food. The fungus remained after ten days of daily flipping into fresh vials. A sample taken from the surface of the food in one affected vial and spread onto fresh food without flies confirmed my suspicion of fungus. 
Does anyone have any tips or should I give up on this line?
Thank you for any help!
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Removing fungus from contaminated stocks is very tricky.
If you have flies from vials which do not have fungus, I would recommend to start a new culture from non-contaminated flies and discard the one from the contaminated vial.
If you don't have any non-contaminated vials of a certain stock, then collect some newly emerged males and females. Under the microscope brush off the fungus (as much possible). And transfer these flies to a new vial. 
You can also pre-treat the food with 50%vinegar ( by adding it on the surface of the food and letting it dry) before adding the parents to the vial. Brood the vials on day 7.
Another method is that collect the eggs (embryos) which can be gently treated with 50% bleach (as we do before embryo injections) and transfer them to new vials. Some will die but enough will survive to start the new contamination free culture.
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I am trying to find a recipe to add 2 deoxyglucose food for Drosophila. In Munica dus et al., 2011 paper, they have mentioned feeding flies with 400mM 2deoxyglucose but I wonder how you make it?Is it just on a plug and feeding them on that plug?or adding that to food?
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According to the paper you mention, they appear to add glucose directly to the water and agar solution before allowing it to cool and harden. You can also find similar agar/glucose recipes with fruit juices in the attached article. 
Another recipe to consult:
Yeast-glucose medium for Drosophila (Cold Spring Harbor Protocols)
30g agar, 100g dry yeast, 100g glucose, 15ml Nipagin M (10% [w/v] in 95% ethanol) (Nipa Laboratories Ltd.)
Mix the three dry ingredients in a blender and add tap water to 1 liter. Bring to a boil slowly and simmer to thicken. Then add the Nipagin M (fungal inhibitor).
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Hi guys! I want to produce some dsRNA to transfect my Drosophila S2 cells. I have been searching and there are a lot of kits available. Do you recommend any kit in particular?
Thanks in advance!
Patrícia
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I have used MEGA script® RNAi kit ( Ambion Applied Biosystems, Austin, TX, USA) for Synthesis of dsRNAs. it worked perfect.
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I am interested in measuring Acetylcholine and Glutamate neurotransmitters from Drosophila melanogaster heads. Does anyone have a experience with that? Could someone help me with protocols or any other tip?
Thank you
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   Hi Breda,
   Recently we mensuared the acetylcholinesterase activity (data under analysis for publishing), easier than HPLC...So, if you wish, I can forward it when published.
   Best,
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Specifically, does long term red light exposure (~12-24 hours) influence post exposure vision? I'd like to do some visual attention assays following optogenetic activation, however, my controls (not provided all-trans-retinal) are performing abnormally in visual behavior assays, which suggest that the red light is having a secondary effect.  
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Hi Chelsie,
I have no experience investigating fly vision or long-term red light exposure, but I have used CsChrimson extensively over the past 2 years, both in explant brains (preferably) and in vivo, and might have some clues to what you're seeing.
First of all, my colleagues and I all see some Chrimson activation also in non-retinal fed controls, presumably because the intrinsic levels of retinal in the fly are sufficient to drive Chrimson to some degree. Looking through my data, the non-retinal controls are sometimes responding up to about 50% of those fed retinal (this is GCaMP6f response of downstream neurons).
Secondly, flies do see the red light (I use 630nm). It is sometimes suggested that flies can't see far-red light, presumably because the rhodopsin sensitivity doesn't seem to reach much further than 600nm (see Salcedo et al., 1999, JNeurosci), but my colleagues and I have indeed found subtle behavioural as well as neural responses to light >600nm. Presumably Rh6 still has sufficient sensitivity at 630nm if exposed to light with high enough intensity.
So, to sum up, your effects could come from residual Chrimson activity in your controls, or from the light itself. Have you already tested the genetic controls not expressing Chrimson (or any other red-shifted channelrhodopsin, for that matter)?
Cheers,
Oliver
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I have used GMRGAL4-Aβ42(Human)/CyO fly for detection of amyloid plaques in adult fly brain by using Thioflavin S dye (Sigma). But I didn’t get ant significant results till now. I Repeated it 3 times. I followed the procedure which is mentioned in www.pnas.org/cgi/doi/10.1073/pnas.0909314107 with little modification TS (Sigma) staining was performed to detect fibril Aβ42 deposits . Fly brains were fixed in 4% paraformaldehyde and permeabilized by 2% triton. Brains were then transferred to 0.1 % TS in 50% ethanol for 1 night and destained for 10 minutes in 50% ethanol. After three washes with PBS, they were mounted using DABCO. In the attached Figure, Green dots indicating apoptotic cell stain by thioflavin S. I did not get this type of result.
I scanned it by using confocal microscopy (leica) on Dry lens 20X and oil immersion lens 63X , zoom : 2,3,5
Which steps I have to improve for Thioflavin S staining?
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No Sir @Ridwan Ibrahim 
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Which membrane protein fractionation kit used in Drosophila S2 cell would you recommend ?
Besides, does anyone know that which fraction "nuclear membrane proteins" exist in ? membrane fraction or nuclear fraction? or both of them? 
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I think that for nuclear membrane proteins you should use an extraction method suitable for membrane proteins (as it we give you proteins from both the outer membrane and the nuclear membrane).
I'm afraid I've never used a kit for this type of extraction..
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I would like to stain VNC motor neurons. and their neurites. In looking for antibodies online, I've only found some for the mammalian vGlut (http://www.abcam.com/vglut1-antibody-ab77822.html), but these are not confirmed by the company to work in drosophila. I assume the proteins are similar, but unsure. The only uses of a DVGLUT (drosophila vGlut) antibody I can find are all self-made, which I don't have the resources to do (http://www.sciencedirect.com/science/article/pii/S1567133X05001006).
Does anybody here have experience with this?
Thank you
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Hi Alexander, 
We have in the past obtained the antibody you mentioned from Mahr and Aberle, and can confirm that it works really well in the Drosophila brain. 
I'd be happy to send you an aliquot but I suggest you write to Hermann Aberle directly first. 
Happy staining! 
Oliver 
Edit: I should also add that I've tried several Abcam mammalian antibodies in the Drosophila brain in the past, without any success whatsoever... 
Edit2: It's also worth adding that in general in the Drosophila community, people are very happy to share antibodies with other researchers. I've already obtained several ABs by writing to authors that created these, in some instances in the 1990s, and they were promptly sent and always worked great. 
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Hello, 
I am trying to prepare a lethality curve for irradiation dosages in the Drosophila melanogaster. Far as I understand, I should take 3rd instar larvae and treat them with increasing doses of x-ray and then count how many eclose into an adult vs. dead as part of the survival % for each dose. 
Is there a specific protocol I can follow to create the lethality/survival curve? I have read some papers what to expect but details about how to set up the protocol or the best response curve measurements would help. 
Thank you,
-J
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Thank you Charles, this is a great start for trying to develop criteria for a new protocol. I will try to adjust mortality assessment for irradiation. 
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Fungal contamination is very much dependent on the activity of larvae. Transgenic lines often have low fecundity as a result of which they also have low laraval activity. In this case you would get loads of fungal contamination even of your native strain is working fine. I would suggest you to use a cage (usually used for collecting embryos) and transfer 2nd instar larvae in fresh vials. Once you have enough, transfer them to bottles. This way you can get rid of fungal contamination. Flies carry spores so everytime you are transferring now you are also transferring fungus spores. Native flies show activity, hence no fungus. Transferring larvae would eliminate spores.
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Out lab has a stock of flies with our favorite balancer genes in it that we use all the time, and we've been having a problem with it where the flies will die in their pupae just before eclosing. We have not had this problem in the past, nor are we seeing the issue in any of our other stocks. Does anyone know why this happens and what we can do to fix it?
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Hi Jessica, 
the pupal lethality could be due to infestation with bacteria/fungi on the surface of the larvae or in their guts. Alternatively, some balancers do not prevent recombination completely ( for example at the tip of the chromosome). In these cases, they accumulate potentially lethal mutations over long periods of time. Even the mutations might be not recessive lethal, they can contribute to reduced fitness. 
There are food compositions more prone to the growth of unwanted germs. I would let the larvae grow till stage 3 and wash them out of the food (1x PBS with a restrainer). I would put them for the last day in a fresh vial with living yeast before pupariation. This reduces the germ load. Use a small time window to collect the eggs so the larvae  are of the same age. Pick the biggest larvae.
Try to raise them on a food with a slightly enhanced amount of propionic acid, Nipagin or a combination of both. In severe cases try Pen/Strep. 
If this fails it is likely due to the accumulation of some mutations. Then ask around in the fly pusher community if someone has a similar/same stock and get it from them (beware of mites!!!). 
You can reconstruct the stock by crosses  but that can take some time especially for double balancer strains. 
I hope this helps a bit. 
Kind regards 
David 
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Please, can somebody help me, our group works with drosophila melanogaster, our stock seems infected, a large amount of mucus is present in the standard medium and the abdomen of flies is swollen, even the locomotion is affected. Has somebody experienced a similar problem? Is there a manner to clean the stock? Thanks 
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Thank
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Hi all,
I'm studying a tiny tissue in Drosophila larvae, which only has about 50 cells on average. I want to measure cytosolic aconitase. Commercial aconitase assay kits require about 106 cells on average for a reliable experiment. So I wonder if any of you have any suggestion of a proper protocol or tissue collection tips or any other idea.
Thank you in advance.
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Yoou can pool samples that belong to the same treatment groups to get the number of cells required.
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I would like to know, is there any marker available which can distinguish between Go and G1 cells in Drosophila tissues. 
Thank you.
Regards
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These articles may be helpful
Dynamic Control of Cell Cycle and Growth Coupling by Ecdysone, EGFR, and PI3K Signaling in Drosophila Histoblasts, Nikolay Ninov,Cristina Manjón,Enrique Martín-Blanco PLOShttp://dx.doi.org/10.1371/journal.pbio.1000079
Biochim Biophys Acta. 2007 Apr; 1772(4): 446–456.  doi: 10.1016/j.bbadis.2006.10.007
Connecting cell-cycle activation to neurodegeneration in Drosophila
Vikram Khurana and Mel B. Feany
,
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fixation with 2% gluteraldehyde and 2% formaldehyde has been tried
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Thank you Monalisa :)
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What is the best alternative of protein hydrolysate for rearing fruit flies?
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Hello Dear
Three protein sources (yeast Saccharomyces cerevisiae) were evaluated in three treatments: T1 = yeast Bionis YE MF® (Biorigin, Lençóis Paulista, São Paulo); T2 = yeast Bionis YE NS® (Biorigin, Lençóis Paulista, São Paulo) and T3 = yeast YHE (Yeast Hydrolysate Enzymatic) (MP Biomedical LLC, Solon, Ohio, USA). The treatments T1 and T2 were alternatives protein sources to the imported yeast (T3). In addition to protein, the diet had wheat germ and sugar (proportion of 1:1:3, respectively) to provide the complete recipe.
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Anuário Brasileiro de Fruticultura. Gazeta, Santa Cruz do Sul. 2009.         [ Links ]
Buena LD, Dueñas RG. Avances sobre la cria artificial deAnastrepha fraterculus (Wied.) (Diptera:Tephritidae) en Colombia. In: Proceedings of a workshop organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. Viña del Mar, Chile. IAEA-TECDOC-1064; 1999. p. 85-94.         [ Links ]
Chang CL. Effect of Amino Acids on Larvae and Adults ofCeratitis capitata (Diptera: Tephritidae). Ann. Entomol. Soc. Am. 2004; 97: 529-535.         [ Links ]
Chang CL, Vargas  RI. Wheat germ oil and its effects on a liquid larval rearing diet for oriental fruit flies (Diptera: Tephritidae). J. Econ. Entomol. 2007; 100:  322-326.         [ Links ]
Chang CL, Albrecht C, El-Shall SSA, Kurashima R. Adult reproductive capacity of Ceratitis capitata (Diptera: Tephritidae) on a chemically defined diet. Ann. Entomol. Soc. Am. 2001; 94: 702-706.         [ Links ]
Costa KZ, Faggioni KM, Lima FTW, Kamya AC, Rocha ACP, Morelli R, et al. Criação de Anastrepha obliqua Macquart, 1835 (Diptera:Tephritidae) em dieta artificial In: CD Resumos. Simpósio científico dos pós-graduandos no Cena/USP, Piracicaba, 2010.         [ Links ]
Dadd RH.  Nutritional organisms. In: Kerkut GA, Gilbert LI. Comprehensive insect physiology biochemistry and pharmacology. Volume 4, Pergamon Press. 1985. p. 313-390.         [ Links ]
Dewey EM, McNabb SL, Ewer J, Kuo GR, Takanishi CL, Truman JW, et al. Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading. Curr. Biol. 2004; 14: 1208-1213.         [ Links ]
FAO/IAEA/USDA. Manual for product quality control and shipping procedures for sterile mass-reared Tephritid fruit flies, Version 5.0, Vienna: IAEA; 2005.         [ Links ]
Gilmour D. The biochemistry of insects. New York: Academic Press;1969.         [ Links ]
Hendrichs J, Vreysen MJB, Enkerlin WR, Cayol JP. Strategic options in using sterile insects for area-wide integrated pest management. In: Dyck VA, Hendrichs J, Robinson AS.Sterile insect technique: principles and practice in area-wide integrated pest managemen. Dordrecht: Springer; 2005. p. 563-600.         [ Links ]
ISPM 26. Establishment of pest free areas for fruit flies (Tephritidae). Rome: IPPC, FAO. 2006.         [ Links ]
Jaldo HE, Gramajo MC, Willink E. Mass rearing ofAnastrepha fraterculus (Diptera: Tephritidae): a preliminary strategy. Fla. Entomol. 2001; 84: 716-718.         [ Links ]
Kaur S, Srivastava BG. Effect of B-vitamins on various parameters of reproductive potential of Dacus cucurbitae(Coquillett). Indian J. Entomol. 1991; 53: 543-547.         [ Links ]
Klassen W, Curtis CF. History of the sterile insect technique. In: Dyck VA, Hendrichs J, Robinson AS.Sterile insect technique: principles and practice in area-wide integrated pest managemen. Dordrecht: Springer; 2005. p. 3-36.         [ Links ]
Lance DR, McInnis DO.  Biological Basis of the Sterile Insect Technique. In: Dyck VA, Hendrichs J, Robinson AS.Sterile insect technique: principles and practice in area-wide integrated pest managemen. Dordrecht: Springer; 2005. p. 69-94.         [ Links ]
Luo CW, Dewey EM, Sudo S, Ewer, J, Hsu SY, Hener Gger HW, et al. Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2. PNAS, 2005;102: 2820-2825.         [ Links ]
Malavasi A, Zucchi RA, Sugayama RL. Biogeografia. In: Malavasi A, Zucchi RA. Moscas-das-frutas de importância econômica no Brasil: conhecimento básico e aplicado. Ribeirão Preto, Holos Editora; 2000. p. 93-98.         [ Links ]
Malavasi A, Nascimento AS, Paranhos BJ, Costa MLZ, Walder JMM. Establishment of a Mediterranean fruit flyCeratitis capitata, fruit fly parasitoids, and codling mothCydia pomonella rearing facility in north-eastern Brazil. In:  M.J.B. Vreysen MJB, Robinson AS, Hendrichs J. Area wide control of insect pests: from research to field implementation, Dordrecht: Springer; 2007. p. 175-182.         [ Links ]
Ortiz G. Introduction. In: Proceedings of a workshop organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. Viña del Mar, Chile, IAEA-TECDOC-1064; 1999. p. 1-2.         [ Links ]
Parker AG. Mass-rearing for sterile insect release. Biological Basis of the Sterile Insect Technique. In: Dyck VA, Hendrichs J, Robinson AS.Sterile insect technique: principles and practice in area-wide integrated pest managemen. Dordrecht: Springer; 2005. p. 209-232.         [ Links ]
R Development Core Team. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2009. [accessed  on Jun 2011] Available from: www.R-project.org.         [ Links ]
Salles LAB. Metodologia de criação de Anastrepha fraterculus (Wied., 1830) (Diptera: Tephritidae) em dieta artificial em laboratório. An. Soc. Ent. Brasil,  1992; 21: 479-486.         [ Links ]
Salles LAB. Rearing of Anastrepha fraterculus(Wiedemann). In: Proceedings of a workshop organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. Viña del Mar, Chile, IAEA-TECDOC-1064; 1999. p. 95-100.         [ Links ]
Tsiropoulos GJ. The importance of vitamins in adult Dacus oleae (Diptera:Tephritidae) nutrition. Ann. Entomol. Soc. Am. 1980; 73: 705-707.         [ Links ]
Vera MT, Abraham S, Oviedo A, Willink E.  Demographic and quality control parameters of Anastrepha fraterculus(Diptera: Tephritidae) maintained under artificial rearing.Fla. Entomol. 2007; 90: 53-57.         [ Links ]
Walder JMM. Produção de moscas-das-frutas e seus inimigos naturais: associação de moscas estéreis e controle biológico. In: Parra JRP, Botelho PSM, Corrêa-Ferreira BS, Bento JMS. Controle Biológico no Brasil: Parasitóides e predadores. São Paulo: Manole; 2002. p.181-190.         [ Links ]
Whitten M, Mahon R. Misconceptions and constraints. . In: Dyck VA, Hendrichs J, Robinson AS. Sterile insect technique: principles and practice in area-wide integrated pest managemen. Dordrecht: Springer; 2005. p. 601-626.         [ Links ]
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Hello. Is there any protocol or video related to dissection of Drosophila motor neurons in adult flies.
Thank you
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Thank you so much
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I am looking for wild flies that have been recently caught. The fewer generations they've already been in the lab the better, definitely < 10. If you have a line that you're willing to share, please let me know!
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We are going to collect flies during mid October from Leicestershire UK. I'm happy to share. But you need to get a license for getting flies into the USA.
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The GeneX is lethal in homozygous state. I crossed GeneX/Cyo x GeneX/Cyo only to get adult non CyO flies post drug treatment to reverse the GenX function. How to ascertain these flies are GenX homozygotes and without the Cyo balancer. I want to do a PCR with GeneX and CyO primer sets. But i cannot find CyO nucleotide sequence. 
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Dear Hitoshi, I do not have a marker on the balancer chromosome. Is there any other way to identify the presence of Cyo Balancer?
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Hi all! I'm about to start working on Drosophila's brain and as it turns out, the lab doesn't have the right forceps nor other tools to dissect the brain, which brand and size would you recommend me to use?
Thank you so much!
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Dear Marcela Cárdenas-Tueme 
please check the attachment  and i do agree with Jonathan M Blagburn
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Dear all,
We are trying to investigate some behavioural tests on the spotted-wing drosophila. Anyone knows how we can distinguish between the winter morph and the summer morph? Thanks in advance,
Best regards,
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Dear Ammar did you know these papers?
Toxopeus J et al. 2016. Reproductive arrest and stress resistance in winter-acclimated
Seasonal cues induce phenotypic plasticity of Drosophila suzukii to enhance winter survival. Shearer et al., 2016.  BMC Ecol (2016) 16:11 DOI 10.1186/s12898-016-0070-3
Hope it helps.
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So I am trying to do ISH on adult drosophila testis and ovaries. So far I have the procedure down until the last part on day 3 where I am doing the developing/enzymatic reaction and the mounting. Currently the protocol calls for me to transfer from a tube into a dissecting disc well. I can do this transfer fine. The next step is adding the reaction solution, waiting, then washing with a few different things, and afterwards adding GMM (Canada balsam:methyl salicylate) to the wells and then taking that and putting it on a slide to mount. My difficulty is that the testis and ovaries are sticking to the glass in the dissecting well and when i try to dislodge them many end up disintegrating. As such from starting with 50 in a well i ended up with 3 on the slide. I am hoping to increase the success rate on this. 
To that end I think I have a solution and would like to run it all by the community here for feedback and suggestions. I am proposing taking a poly lysine coated slide and using a grease/wax pen to draw a barrier and make a mini well. I would then add the testis directly into this mini well and perform all the reaction and washes within it. My reasoning is that since my main problem is that the testis are sticking to the glass and disintegrate on contact of when being disturbed. If i put them directly on the slide I should be able to use this weakness as a strength. What is everyones thoughts on this? 
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Hello everyone, sorry for the wait. I can confirm that using the wax pencil worked wonderfully, even with a non-coated slide.
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none 
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Hi Avnika
I attach a handbook related with estimation of protein concentration. I hope you find it useful
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I am trying to do a ROS assay in Drosophila larval brain using 30uM DHE. But I have no reference image to compare to. Also, since the sample is too thick, most of the staining is superficial. How can I troubleshoot this?
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Dear Sourajit Das!
You can try to do embedding process in fresh tissues and reduce the concentration of staining, inhibition time and do one more time for destining process. i will be good observation... all the best
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Hi I'm working with Drosophila guts and am looking to see if and where cells are undergoing apoptosis. I am trying to understand how TUNEL works to use that as an assay for apoptosis detection. I am having a hard time understanding what I'm ordering and the protocol involved. Is TUNEL is part of the cell detection kit by roche or is it a seperate thing? What's the difference between the kits TMR and Fluorescein, does it matter? How does the tissue need to be prepared, and I understand TUNEL labels the cleaved DNA in apoptotic cells, but does it do that with fluorescence that can be imaged or is there another step after that that needs to be done to view under a microscope? Are there any papers that anyone knows of that can describe the protocols used? Thank you do any of the answers to these questions
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We used the UAS-CD8-PARP::Venus reporter staining with cleaved PARP (cPARP) as described in Fig.3 to assay apoptosis in the midgut. We have had bad experiences with trying to use TUNEL in the fly intestine. I would not advise using it in flies, since we had either no signal (also with + controls expressing Reaper that should trigger apoptosis), or too much signal with any of the TUNEL-based methods. Suijkerbuik et al., http://www.cell.com/current-biology/fulltext/S0960-9822(15)01574-2 also used this in the midgut to address apoptosis. The antibody data is in their supplementary. Hope this helps!
Jerome
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I am interested ideally in a fluorescent TORC sensor, or indeed in any other way to obtain a TOR activity readout in fly guts. Please advise.
Thank you!
Best,
Lena
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There's a good anti-phospho-4EBP antibody described for gut immunostaining (Cell Signaling #2855) that is high in EB's with high Tor activity. Hope this helps!
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What is the signature of the calcium regulating protein? Are there any in vivo reporters to monitior caicium influx?
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Hey buddy, you can use calcium indicators like GCaMP6 (UAS-GCaMP6) and do a live imaging to monitor the calcium influx in vivo.
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I want to evaluate whether some drugs increase lifespan of a Drosophila disease model. However I am confuse of which is the best statistical test for this experiment. Is Kaplan-Meier a good method, if so which cut off do you recommend me to use? Or is it better to use other methods such as Gompertz or Cox regression?
Ask
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Thanks Frederic, I read a lot more and I think I understand the analysis uses and limitations. The only think that still puzzles me is that I had 3 repeats for each treatment (2 different treatments, with 3 vials each repeat, inside each vial I have 20 flies). At the beginning I thought to make an average of the Km survival of the three repeats per treatment. Plot it with error bars and do the statistical analysis. But know I am thinking I could also treat each fly as an independent subject (as long as I have the same treatment treatment) regardless the vial they were in, as this makes more sense for using the survival analysis. My rational to do this is to think as if this was a Phase III clinical trial performed in different countries, but I don't know if this makes any sense at all.
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Dear all, I would like to bring some Drosophila strains from Japan to another country (Indonesia) with me on the plane. Some of the flies are mutant (lacking for some proteins related to immune system). I have ask some institutions/authorities in Indonesia in regards to this matter but they did not give me a proper answer. Perhaps someone had the same experience and would like to share?
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Thank you for your kind suggestion Antara Das. I will try to do so and hoping this won't create a fuss to Immigration officer when I arrive in Indonesia. :)
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I am looking for a method that is
  • inexpensive as possible
  • has good discrimination
  • can be used with few flies at once
I have seen several different methodologies in the literature, but it isn't clear from that how they would meet these criteria.
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There are a few different respirometry methods you could follow; it depends what exactly you need from these measurements. Flow-through is typically more expensive but also more stable and reliable than closed box at the scale of an individual fly. In any case it is likely to be relatively expensive to set up, as to achieve good discrimination between treatment groups or individuals, and because the animals output such a small volume of CO2, the CO2 analyser precision must be high. Fortunately, D. melanogaster have a respiratory quotient of ~1 so you do not need to also measure O2 production to accurately calculate metabolic rate. You only need a stable baseline reading and then a measure of CO2 production to estimate metabolic rate at rest, but you might be limited to the higher end of Sable Systems or Li-Cor instruments in order to reliably detect a difference between baseline and animal CO2 rates.
Some examples of Drosophila respirometry:
Berrigan and Partridge (1997) used a flow-through respirometry on individuals using a combination of Sable and Li-Cor instruments.
Promislow and Haselkorn (2002) used a constant volume respirometry system with a multiplexer (Sable) to measure multiple flies at once.
I have used a two-channel Li-Cor flow-through respirometry system to measure individual flies, the methods of which are published here:
Hope this helps, and I can try to answer any further questions you may have.
Pieter
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I am working on 30um sections of adult drosophila brains. I dual stain the sections first with Alexa-fluor 594 anti-HRP stain and then with DAPI to stain the neurons and the nuclei respectively. 
Right now, after (cryo)sectioning, I capture the sections on Superfrost GoldPlus slides, add few microliters of the staining solution onto the slide and cover it with parafilm for uniform spreading of the stain. I do the same procedure for washing with PBS, however, the sections get untethered from the slides 
1. When peeling the parafilm away, the section gets stuck to the parafilm (or)
2. The section begins to float while washing with PBS or adding the staining solution.
How do I prevent this from happening? 
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Hi Rathnavel, 
Did you verify if your slides are good? We are using pre-cleaned Superfrost Plus slides and the quality can differ from lot to lot. Also instead of Parafilm you might want to consider using a PAP - pen to outline your sections, to keep the solution on the sections. 
Best Simone
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Specifically, I need to quantify cell differentiation event in wing imaginal disc of 3rd stage larvae. Is this product "Cell Signal Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) " compatible ? 
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SILAC stands for Stable Isotope Labeling of Amino Acids in Cell Culture. We want to SILAC label flies and then later on use them for our study. However, the method of labeling is very demanding. Therefore, we are looking for some source/company from which we could order pre-made SILAC fly food.
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have you brought food already?
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This answer could go into molecular details and/or details of the breeding experiments carried out to establish the genetic basis of eye colour.
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I am looking for a protocol for dissecting Drosophila flight muscles does anyone have one?
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Dear Andrew,
Attached is a protocol for dissection of the ventral nerve cord of Drosophila. I have read the protocol and believe that it can serve as a protocol for dissecting Drosophila flight muscles.
Hoping I am right,
Rafik
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How do I test if mate success is dependent on males or the female? 
We wanted to investigate how certain mutations affect male mating success. 
Male conditions (4 total):
1.) mutant 1
2.) mutant 2
3.) wild type 1
4.) wild type 2
Female Conditions (3 total):
1.) mutant 1
2.) mutant 2
3) wild type 
Methods: 
Two males of different mutation conditions (see above) are placed into an arena with one female of a specific mutation status. The male that successfully mates with the female is given a score of 1. The loser receives a score of 0. 
Examples of some combinations we might test:
Female (Mutant 1) x Male (Mutant 1) x Male (Mutant 2)
Female (Mutant 2) x Male (Mutant 1) x Male (Wild Type 1)
Female  (Wild Type) x Male (Mutant 2) x Male (Mutant 2)
.. etc. etc. until all possible combinations have been explored (14 combinations in total)
I have already run the pairwise comparisons between the males and it is fairly obviously that the mutations are influencing mate success (ie some mutants mate more successfully than other), however I still have to control for effects of females. 
What statistical test can I preform to rule out the possibility that mating success is based on female mate preference and not male mating behaviour? 
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Hi Chelsie,
It is very difficult to decipher if male mating success is due to male behaviour or female choice, specially in Drosophila.
 One way(the more straightforward one) of figuring it out is if you have data on courtship behaviour, i.e., if you can show that your mutants are courting more/less than wild types when provided with the same female (sampled from the same gene pool). (see: http://www.sciencedirect.com/science/article/pii/S0003347287802816)
Since you collected data on male mating success in the way mentioned above, you could also calculate effect sizes and show that the extent of mating success does not change even if the female is changed. For example, let's take two scenarios:
Female (Mutant 2) x Male (Mutant 1) x Male (Wild Type 1) 
Female (Mutant 1) x Male (Mutant 1) x Male (Wild Type 1)
Now if in both the cases Male(M1) has roughly the same proportion of mating success, then you could say that at least part of it is due to male mating behaviour. It's more of a roundabout thing, but if the data are clean, it should work.
Hope this helps!
Regards,
Zeeshan
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Expression patterns of Ilp2 and Ilp5 genes in Drosophila melanogaster under fed and starved conditions.
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Thank you very much Marc!!
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The multiscale issues I got so far are as follows:
-- proliferation & differentiation of cells
-- the evolution of multiple cells
Are there any other spatial multiscale issues or temporal multiscale issues ones? Thanks a lot.
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please refer the following link
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I need a method to freeze flies for molecular analysis while they are in a behavioral sleep assay. Specifically, I need to snap freeze them while they are in deep sleep without arousing them. Individual flies will be assayed in typical trikinetics glass activity tubes.
Any suggestions, even if a little out there, would be most appreciated!
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Hi,
how are u planning to ensure that they are in deep sleep? Will the flies be under recording in DAM system when you wish to freeze them? or will you load them into glass tubes, place on a horizontal surface and visually observe them to check for activity in a given window (say 5-15 min ) to decide their sleep state? I am not sure if I can help you if you want them to be under recording when u wish to freeze them. Otherwise I guess there is a way. 
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i strictly want a nuclear localised antibody. thank you.
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Hi Jayesh,
Could you possibly use antibodies against epithelial markers that are not nuclear-localized for GTRACE?  I ask because most of the reliable epithelial specific antibodies that I have used, e.g. those against the septate junction protein Coracle, tend to be membrane specific.
A possible alternative would be using FISH to hybridize transcripts for epithelial markers.  It wouldn't be quite as easy or as sensitive for GTRACE-ing, but might get the job done.
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 what will be the effect of mating on longevity ? please include any link if u can for the longevity graph in these two cases. thank you.
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Females live much longer than males. Most paper used virgin female in longevity experiment. So you can not mix female and male together in your experiments.  It will not make sense to your data.
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Hello. I want to check the expression pattern of a particular protein after transfecting it into S2 cells and then performing whole cell extraction. I need some suggestions regarding how to perform whole cell extraction using S2 cells. Previously I tried just to lysate the cells using 4X sample buffer and then boil it at 98 degree celsius for 5 minutes. It seemed that the expected band is not so prominent as I hoped it to be.
Any suggestion would be very helpful. Thanks in advance.
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We would like to make RNA-seq libraries from Drosophila RNA. It is important that we deplete rRNA rather than enrich for Poly(A) as we would like to analyse some non-polyadenylated species in our datasets.
I have read that the Illumina Ribo-Zero rRNA depletion kit (Human, Mouse, Rat) is reasonably effective, although does not fully remove 5S or 28S (right arm) rRNA.
I'm also thinking about an rRNA "RT blocking" rather than "depletion" strategy a la the method presented in the attached paper, although I would have to devise a strategy for removing all types of rRNA (not just 2S).
Has anyone had experience with this, and would you please share some wisdom? What sort of parameters would I have to be careful to consider using these approaches? Do you have alternative suggestions?
As an aside, these RNA-seq libraries are likely to be from low-input RNA, and the quality could be less-than-perfect as we are isolating RNA after formaldehyde fixation.
Thank you in advance for your intelligent answers.
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Hi all,
Apologies for the radio silence!
It turns out that the homebrew biotinylated probe depletion works a treat (with some caveats).
The steps:
1) Design PCR primers to amplify rDNA genes from genomic DNA. You want to cover basically the whole RNA sequence. For longer species - 18S and 28S (L/R) - we amplified two halves of each gene (so, two probes for 18S and a total of four probes for 28S)
2) In vitro transcribe your rDNA PCR products by incorporating Biotin-UTP into the mix. There are plenty of different ways to do this - choose your favourite
3) Quantify your RNA probe concentration using Qubit and pool your probes so that there is at least a 5x excess of probe:RNA (this requires optimisation, sorry)
4) Hybridise your probe mix to RNA sample, followed by incubation with strepavidin-coated magnetic beads.
5) Keep supernatant! This is your rRNA-depleted sample.
Each step requires optimisation. I found that the IVT (despite being quite a routine procedure) was quite temperamental. You can assess depletion using qPCR against rRNA species - I find this is much more quantitative and informative than Bioanalyzer analysis.
NOTE: you may have to tinker with the probe pooling concentrations. I found that if I add too much excess that the beads don't completely remove the probe and then when you do the qPCR it looks like the rRNA levels have actually increased!
CAVEAT: I had success removing all species but 5S. This is the same as Ribo-Zero. Not something I would lose sleep over as 5S is smaller than most library prep size cutoffs (usually 200nt+). Maybe you will have more success?
However, after all of this, I have all-but-abandoned this protocol for RNA-Seq. We are currently using a kit from NuGen (Ovation RNA-Seq System for Drosophila - http://www.nugen.com/products/ngs/ovation-rna-seq-systems-1-16-model-organisms) that is VERY reasonably priced per reaction and has a nice "rRNA exclusion" step where - rather than depleting - it excludes unwanted RNAs from PCR amplification in the final step. It's very nice, and in our first samples we saw between 1.5-5% rRNA in our final sequencing libraries. Plus, you can customise it to exclude other contaminating RNAs.
If anyone has questions, feel free to shoot me an email at alexandra.mccorkindale@mdc-berlin.de
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I know several gene mutations in which eye color changes in drosophila melanogaster but I could not find the mechanism underlying yellow eyes ((RB model of drosophila Melanogaster) in melanogaster.  I will be glad if someone could explain it to me.
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Dear Saman,
Attached is a chapter that might help you.
Good luck
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Somewhat is already know about D.suzukii overwintering in invaded areas, but what about aestivation? In South Mediterranean areas trap captures usually disappear (or almost disappear) during summer months.
Where is the fly? Anyone has recorded local migrations or refuge areas as was reported for Lepidoptera?
What do you think? Thanks in advance
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The conclusion we came to while monitoring for D. suzukii was that during the summer months, traps were not as attractive as the fruit that was readily available, giving the impression that the flies were not present (this was monitoring done in blueberry fields using apple cider vinegar; this may not be the case with other baits/lures that have proven to be more attractive).
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I have been raising "winter morphs" of Drosophila suzukii by exposing larvae at 5 deg. C for 2 - 4 weeks under totally dark conditions. The adults are very dark in pigmentation; however, none of the winter morph males have their usual wing spots. One specimen had just one wing spot on one wing. All other winter morph males were "spotless". The summer morph flies that emerged from the same cultures all had the usual apical wing spots. Is this normal? or does it take longer for "winter morph' flies to develop their wing spots? I included a picture. The winter morph males are in the upper left, summer morph males are in the upper right group. Corresponding females are below  Hope you can help. Thanks in advance. Blair
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Hi Blair,
I've been rearing D. suzukii at 16°C for full cycle, and all the individuals showed signs of winter morph (i.e. thoracic pigmentation, longer wings etc.) but all the males had spots. Even some females showed slight shades of pigmentation around the apical part of the wings when looked under a binocular.
At what age were the male when you took this picture? Sometimes the spot takes a few days to appear, might be the reason...if not, very interesting!
Best
Antoine
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I would like to try and stabilize a protein that is tagged with FLAG tag extracellularly with the help of anti-FLAG anitbody. Such experiments have been done in cell culture, but I have to do it in the drosophila embryo. Has anyone tried this and is there another way of stabilizing an otherwise stable proteins (except genetically engineering them to have dimerization domains).?
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I am a theorician and would like to test the role of the centrosome in positioning planar cell polarity components: Dsas4 mutants lack centrioles and centrosome after third larval instar, and by my theoretical model wing cells cannot dispose correctly PCP components: I am looking for a Drosophila lab researcher who helps me to experimentally test this hypothesis.
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Hello Marco,
Since Sas4 mutants are viable I do not think that the main function of centrosomes is to establish a 3D spherical polarity. Apical-basal polarity is not affected.
I do agree that their may be a function of centrosomes as MTOC in cooperation with PCP pathways to guide cell movements as observed in the zebrafish (Sepich et al, Development, 2011).
Ciao
Floris
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I'm looking for three dimensional synthetic image data sets that would correspond to epithelial tissues like Drosophila wing disc or Amnioserosa cells, but basically any 3D tissue image set would be helpful. Needs are: it's 3D, mimicking membrane labelled epithelial tissue, simulating Confocal microscopy stacks. And of course the ground truth of how many cells there are to evaluate 3D segmentations.
Thanks in advance
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Contact authors of
Cell. 2014 Oct 9;159(2):415-27. doi: 10.1016/j.cell.2014.09.007.
Interplay of cell shape and division orientation promotes robust morphogenesis of developing epithelia.
Xiong F1, Ma W1, Hiscock TW1, Mosaliganti KR1, Tentner AR1, Brakke KA2, Rannou N1, Gelas A1, Souhait L1, Swinburne IA1, Obholzer ND1, Megason SG3.
or
Elife. 2015 May 6;4:05864. doi: 10.7554/eLife.05864.
MorphoGraphX: A platform for quantifying morphogenesis in 4D.
Barbier de Reuille P1, Routier-Kierzkowska AL2, Kierzkowski D2, Bassel GW3, Schüpbach T4, Tauriello G5, Bajpai N2, Strauss S2, Weber A1, Kiss A6, Burian A1, Hofhuis H2, Sapala A2, Lipowczan M7, Heimlicher MB8, Robinson S1, Bayer EM9, Basler K8, Koumoutsakos P5, Roeder AH10, Aegerter-Wilmsen T8, Nakayama N1, Tsiantis M2, Hay A2, Kwiatkowska D7, Xenarios I4, Kuhlemeier C1, Smith RS1.
or
nadine peyrieras Lab
All have 3/4D skeletonized data of epithelia.
Ciao
FLoris
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Can anyone suggest dyes to sense calcium flux in drosophila imaginal discs?
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I would say that it is quite standard to use the gCAMP lines now. They have been used primarily (and successfully) in neurons, which are a sensitive system in themselves. But you are totally right; I would use multiple methods to validate your experiment.
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I am trying to elucidate the expression patterns of a number of candidate genes in deeper tissues (i.e. the visceral mesoderm and gut) of stage 9-13 embryos, and am currently employing the protocol outlined by Kosman et al. (http://www.sciencemag.org/content/305/5685/846.abstract) for RNA probe generation and subsequent fragmentation, purification, as well as embryo prep, incubation, and immunostaining. So far I have had inconsistent results, which I suspect to be due to inadequate RNA probe concentration and improper penetration of riboprobes into deeper tissues. I have not had any issues with FISH when assaying for genes expressed in the ectoderm, and have found variability when assaying for genes with known expression patterns in deeper tissues (i.e. within the same batch of samples and conditions, the same riboprobe shows a decent expression pattern in some samples and not in others).
Does anyone have any tips for improving riboprobe concentration and permeabilization of tissues? I have used DNA concentrations anywhere between 200ng to 1ug, followed sterile technique to the best of my ability, and varied probe incubation times anywhere between 16-24 hours at temperatures ranging from 56C to 64C. I have also used Proteinase K digestion at the recommended concentration (10ug/mL) for 6 minutes. Any suggestions would be greatly appreciated -- thank you very much in advance.
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To increase probe concentration, we aim to use between 500ng-1ug template DNA and run the in vitro transcription reaction at 18 degrees for up to 5 days. Typically, I run for ~48 hrs
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There's a blip in my staining procedure (I thought I had the correct secondary antibody that I need, but I actually need a slightly different one) and I am currently on the primary antibody staining step. Is it ok to store the embryos in PTw at 4 deg. C after washing the embryos? I can't proceed to secondary antibody staining at the moment.
Thanks!
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I have also kept embryos at 4oC in PBS+0.3% Triton after primary for two weeks and they have still stained beautifully. It is quite robust.
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Detect DNA damage.
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I had answered this question before.
This might also help!
CELL BIOLABS INC.
Many kits are mentioned.
DNA may be modified in a variety of ways. DNA methylation has been shown to be involved with almost every biological process, and global DNA methylation levels may be associated with various disease states. Other DNA modifications such as oxidative DNA damage have been implicated in the development of a variety of cancers including colon, breast and prostate. Mutations to checkpoint kinases can ultimately lead to decreased DNA repair and increased susceptibility to cancer.
Recently oxidative RNA damage has been described in conjunction with a wide variety of neurological diseases including Alzheimer’s disease, Parkinson’s disease, and Dementia with Lewy Bodies.
We have developed quick and reliable assays to detect global DNA methylation as well as various types of RNA and DNA damage.
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Hi, I'm curious how fruit flies can sense the amount of ethanol.
According to lots of precious studies I read, flies are pretty sensitive to the alcohol level in their food. But I cannot find any information that if they have ethanol sensors among olfactory receptors even though they prefer ethanol-containing food, especially in specific conditions. 
Is it possible that 1-hexanol or 1-octen-3-ol receptor can detect ethanol as well?
Or, is there other way to sense ethanol?
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In nature, D. melanogaster depends on  rotten friuts. Naturally they have recruited the adh gene in the genome for their adaptation. The sensary system of the flies are also adapted for their survivility. adh null mutation is present in the genome  in nature. They can not tolerate alcohol. Therefore,  this process is complex evolutionary phenomena.
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Looking for protocol for protein isolation from Drosophila Melanogaster.Isolation of protein from flies?
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Weigh 5-10 flies in a pre-weighed eppendorf tube.    Freeze the flies in liquid nitrogen. This prevents proteases from degrading your proteins and protects the protein conformational state of your enzymes (if needing to assay their activity, use an appropriate buffer).  Transfer them quickly to ice and add RIPA buffer (10ul/1mg of flies) with protease inhibitors (use a mini-cocktail from a company like Roche or ThermoFisher).   Using a hand-held homogenizer, homogenize the flies in ice 6X for 2-3 min each.  Leave it on ice for 10 min to extract as many proteins as possible.   Add 2X SDS loading dye with DTT at a 1:1 ratio to prepare your proteins for running them in the gel (coats them with a negative charge and cleaves the sulfhydril bonds to linearize them).  Boil at 95oC for 5 min (change the temperature and time depending on the hydrophobicity of your protein of interest, i.e. 65oC for 30 min for some membrane proteins).   Good luck! Let me know if you have any problems.      
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What is the best method to isolate Drosophila embryonic hemocytes for RNA-Seq?
What are pros/cons of FACS and MACS?
Any suggestion or protocols, are highly appreciated. Thank you. 
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Time wise, it depends on how many cells you have and what starting percentage you need to start from. FACS takes me about an hour for staining and washing, and then 1-2 hours for sorting.
MACS takes about an hour for staining and each column also 30 minutes, for a total of 2 hours.
Even with the possibility of harming your cells a little I would still prefer FACS because of the superior purity. We have had good results for RNASeq but also hybridization arrays for peripheral blood cells, bone marrow cells, MSC, so FACS is applicable for many cell sources. If you have very large cells, such as MSC, you might want to use a slower sort speed and a bigger noozle (e.g. 100 µm instead of the 70 µm standard)
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I have a mutant Drosophila wherein I suspect that a certain gene has insulator sequences (resembling LTRs) inserted in the region coding for 5'UTR. I wish to search, detect and identify the enhancer of the gene, as well as identify enhancer-promoter miscommunications occuring. Please help.
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Thanks Sudarshana. I actually want to detect the enhancer as well. My first question is WHERE is the enhancer and then I would like to investigate the E-P comm. Well, I hope to benefit from the literature referred by you. Hope to update soon.
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Is there any publication and can be identified at pupal satage
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The really easy way to do it is to sex wandering L3 larvae by identifying the enlarged male gonad. These are ~2/3 down the length of the larvae moving from anterior, presenting as less opaque bilaterally symmetrical 'holes' (also referred to as the 'donut') embedded laterally in the fat body. If these are present, it's a male. If they aren't apparent, it's a female.
Separate these out, place them on Whatman paper  with moistened tissues underneath in petri dishes and let them develop at 25ºC (so you can stage them). In the first instance you can allow them to develop to adults (and score for your efficiency in sexing the larvae). One you're comfortable what you are a 100% accurate then let the sexed/selected larvae develop into whatever stage pupae you are interested in.
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I needed to find the genomic co-ordinates of all drosophila enhancer, robust as well as permissive. Can anyone sent me a link or have any idea how to get to such a file?
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We conducted an experiment and let a bar eye male mate with normal eye vergine female. The results of different members in the group make us confused. I get 4 flies with Bar eyes, my colleague get 20 flies with eyes like what in the attached pictures. we are confused if what is in the picture is bar or normal?  
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I would like to ask 1) Do you know what allele of Bar you are using?  2) Are you working with a balancer chromosome like FM7? 3) Do you have access to the FlyBase website?
If you do, then you can search for Bar on flybase and read about it there. 
The spontaneous mutation Bar1 has a visible dominant phenotype and is the common Bar eye mutation associated with the FM balancer chromosomes. This allele is what Bar was named for and hence it is written with a capital letter "B" which indicates dominance.  It is also true that Bar has alleles (mutations) that are not dominant visible but that are recessive lethal.  This is probably not what you are working with given that you started with a male that is alive with a Bar eye phenotype.    Hope this helps.
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Hi folks,
is there someone familiar with patch-clamp recordings from Dorso Longitudinal flight Muscles (DLM) in drosophila? I'm trying without success yet and every suggestion, papers and tricks are absolutely apreciated. Till now seems impossible to make the seal and thus record something from the fiber. I wish to thank anyone in advance for any help.
Best wishes
Giuseppe
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Hi Miguel, I don't really know the size of the fibers but seeing inthe microscope they seems so tiny. My guess is that is probably better to go through neurons instead muscles because I'm realizing that there more chances and results could be maybe more important. Thanks so much for your help I'll write ya again soon. best whishes
Giuseppe
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In Drosophila melanogaster (fruit flies), how significantly different is the vermilion eye color to the wildtype eye color? I was reading that vermilion has a brighter red pigment than the wildtype flies, but was wondering if vermilion and the wildtype red eye look significantly noticeable. In other words, how much brighter? and are the eye colors different to tell apart as the flies age? 
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Let's say the difference is so "intense" as between purple and dark red. But the difference highly decreases with age. So, to be sure, I suggest to compare the eyes of newly hatched, virgin animals.  If you want to be 100% sure, I suggest to use cho (chocolate) mutant background. Here the vermilion cho double mutant eyes are more like orange compared to wildtype cho animals, which show dark, brownish red eye. We use vermilion cho mutants to make transgenic animals if our transgene bears vermilion+ marker.
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I am interested in obtaining the Illumina RNA sequence for the Drosophila (fruit fly) gut and head. Is there any successful method of preparing the samples?
I would like to know what solution to store the biological samples in, before and after RNA extraction. Also, does 1 fly gut provide enough biological material for the transcriptome analysis, or if I do need more, how many would that be?
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 Jadhav ji,
I am going to use a kit for total RNA extraction and then send it for sequencing. Wouldn't be converting into cDNA myself.
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I need to measure trehalose and glycogen level in Drosophila adults. I've read that it could be done with the use of SIGMA glucose kit in Morris et al., Biochim Biophys Acta. 2012; 1822(8): 1230–1237. But there is more than one kit for glucose detection in SIGMA. I wonder which one I really need.
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