General Dictyostelium Questions

• Who knows about a virus that might infect Dictyostelium ?
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• A big inverted dupliation is found in chromosome 2 in AX2 cells. Does anyone know if there is this kind of duplication (the same or others) in the DH1 cells?
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• Dictyostelium taxomomy: What is the class, order, and family for Dictyostelium discoideum?
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• What is the relationship of the flagellated molds to the core fungi (Zygomycota, Ascomycota, and Basidiomycota)?
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• Does anyone know of a source for a video showing Dicty development?
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• I am looking for some pictures (or better, movies) of dicty cells developing into macrocysts. Do you know of any www address where I could find this?
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• Is there a good reason why Dictyostelium secrete their lysosomal enzymes when they are starved ? Does this play a role in development?
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• Looking for the absence of a gene in the genome.
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• I was wondering who isolated NC4 cells.
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• I could not figure out a way to access up to date primary reads via blast. Is there another web site which I should use?

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Gene Features

• I would like to compare the promoter sequences among a family of genes in Dictyostelium that are differentially developmentally regulated.
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• Dictyostelium gene structure: I am looking for information concerning the prediction of ORFs in Dictyostelium. Of particular interest would be references where the characteristics of the start codon are detailed, as well as predictions of splicing sites.
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• Early developmental promoters for genetic screen.
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• Is TAG a functional codon stop in Dictyostelium genetic code?
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• Terminators: What is the shortest length of sequence that anyone has shown to work as a transcriptional terminator?

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Dictyostelium Transformation

• Transforming two vectors.
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• I want to generate conditional mutants in AX2 strain of Dicty using antisense technique.
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• I'd like to do RNA interference, has anyone a detailed protocol, advices...about this technique.
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• Has anyone in the community tried RNAi by soaking or feeding, as in C. elegans?
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Selection of Transformants

• I am wondering why Herpes Simplex Viral Thymidine kinase- negative selection is not used in Dictyostelium
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• Has anybody used puromycin as selection marker to Dicty?
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• Selectable markers: We would need to obtain homologous recombinants using a resistance marker other than blasticidin.
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• Blasticidin selection on bacterial lawns?
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• I am wondering if somebody has a trick for G418 selection or a good working protocol different from the standard one.
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Transformation Protocols

• Problems with transformation NC4-A4 cells.
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• Transient transfection: Has anyone tried transient expression of a GFP tagged protein in dicty?
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• Has anyone developed a protocol for transient transfection of plasmids or for the introduction of antisense oligos?
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• Can anyone give me a good protocol for electroporation of Dicty in 0.2 cm cuvettes?
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• Does anyone have experience in electroporating fluorescent molecules into Dicty cells?
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• What are the pros & cons of using lipofectamine?
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General Transformation Issues

• Abnormal cellular morphology of transformants
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• We are experiencing a problem about an unstable-lacZ transformant
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• Unstable phenotype

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Gene Disruption

• I am trying to find out the lengths of recombinogenic arms used for generating Dicty knockouts successfully.
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• What are the lengths of recombinogenic arms used in successful KO experiments or what is the shortest reasonable arm length?
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• Need help figuring out suitable KO controls
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• PCR-based gene disruption: Has anyone tried a gene knock out in Dicty using a 60bp oligo nucleotide primer similar to what they use in the yeast system?
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• PCR-based gene disruption: Has anyone working on Dicty has tried making gene knockouts directly with PCR products, as yeast workers now do?
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• In two cases we have now seen (by PCR and Southern blot) that a knock-out strain contains both the wild type band and the knock-out band.
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• While characterizing REMI mutants (made in DH1 cells), we often observed that the insertion of the plasmid (pUCBsrDBam) was accompanied by a deletion of some genomic DNA. Did this already happen to anybody ?
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Gene Expression in Dictyostelium

• Is there an appropriate Tet On or Tet Off expression system for Dictyostelium?
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Dictyostelium as an Expression System

• I would like to know if Dictyostelium would be a suitable expression system to obtain a fully glycosylated product.
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Reporter Genes

• I would like to express a GFP-tagged version of a protein under its own promoter.
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• Does anyone know the half life of GFP expressed in Dictyostelium?
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• Has anyone tried expressing the EGFP form of GFP from Clontech?
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• Does the codon usage of the GFP gene (some of the different types of GFP have been constructed using the human codon usage tables) need to be changed for significant/detectable expression of GFP in Dictyostelium?
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• Has anyone used any of the coloured variants of GFP in Dicty?
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• RFP-ABD
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• Has anyone yet tried hrGFP in Dicty?
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Protein Tags

• Can you suggest a good protein tag for Co-IP in Dicty?
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• I was wondering if there was any consensus on which protein tags work best with Dicty proteins.
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• Is anyone having trouble using myc as an epitope tag? We have tried placing it on two proteins so far and are having no success.
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• Are there any experiences whether N-terminal FLAG-epitopes fused to a protein of interest work well in Dictyostelium and whether FLAG-antibodies can be used for indirect immunofluorescence experiments with regard to background fluorescence?
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• Has anybody used the FLAG epitope in Dicty? Any information on positioning of the epitope, cross reactivity of the antibodies etc would be greatly appreciated.
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• My lab has recently had bad luck with the myc epitope not being visible (by western) when fused at the amino term. of proteins in Dicty. We plan to try an HA tag, and would appreciate any info that anyone has (successes, failures , problems, or other general comments!!!)

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Dictyostelium Vectors

• Where can I find the sequences of Dicty vectors?
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• I am looking for a certain plasmid. Where can I obtain it?
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• I am looking for a dicty vector that enables me to clone a gene of interest at the N-terminus of GFP.
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• Does anyone have a good inducible system for protein expression in Dicty?
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• We would like to overexpress a protein in Dicty. We would appreciate any information with regard to the vectors that are available for overexpression and any other suggestions on methodology.
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• IRES vectors: Has anyone ever tried using internal ribosome entry site vectors?
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• We are looking for an extrachromosomal vector that carries GFP or beta-galactosidase.
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• We are looking for an extrachromosomal vector that we can use to express myosin light chain mutants in Dicty. What is your collective wisdom regarding the best vector?
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• I wonder if anyone can enlighten me on what pREP might be and on some reference on what it is?
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• Has anyone sequenced the thy1 gene that is used as a selectable marker with the JH10 strain?

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Dictyostelium Cell Types and Cell Differentiation

• If we take an aggregate of prespore cells, the posterior part of the slug or assuming we can somehow separate them from the mound, the cells will re-differentiate into 4:1 ratio of psp/pst. Can anyone tell me how long this re-differentiation process takes?
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• Is the stalk cell induction of HM44 density dependent?
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• What is the volume of stalk and spore cells in Dictyostelium discoideum?

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Growing Dictyostelium

• I have a question to people who has isolated Dicties from the nature. I cannot separate the strains from the fungi.

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Media

• Problems preparing SM medium.
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• Problems with HL5 medium.
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• How many of you out there use Formedium HL5? Are you happy?
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• I'm looking for some minimal medium without yeast extract and peptone, in order to prevent interference of these compounds with GFP fluorescence.
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• Has anybody had any success growing Dicty cells in the FM media available through Bio101?
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Growing Dictyostelium in Axenic Medium

• We are experiencing problems in the last months with axenic cell growth. AX2 cells grow well up to 1 x 10E6/ml and then stop growing or grow very slowly
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• I was just reading the the pH optimum for axenic Dicty growth is around 5.5. I don't know the original source of this rumour - does anyone else?
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• Does anyone have tried to set the dicty concentration with OD at 600 nm (or another wavelength)?
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• Is there a simple way to determine the approximate concentration of Dicty cells over time while they are still growing in HL-5 media?
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• Does anyone know how long that dicty cells attached to the dish in HL5 can stay alive at 4 C?

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Growing Dictyostelium on Bacteria

• We are trying to grow dictyo (DH1) on a lawn of E. coli instead of the usual K. aerogenes.
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• I have a question as to which E. coli B/r strain is being used to culture Dictyostelium.
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• I am growing my strains on KA plates, and I want to collect happily growing vegetative cells.
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• Poor growth of Klebsiella on SM plates.
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• Is Klebsiella Kp29 dangerous?

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Molecular Biology Techniques

Genomic DNA Preparations

• Genomic minipreps: Has anyone developed a simple, reliable protocol for performing PCR reactions on Dicty whole cell lysates?
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• Genomic minipreps: Can you recommend a quick and easy method for preparing template DNA from a small sample of cells for doing PCR? I'm looking for a method suitable for screening transformants by PCR.
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• Genomic minipreps: I need a reliable protocol for PCR of cell lysates for use in screening recombinants. Does anybody have a 'quick and dirty' way of preparing lysates for PCR?
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• Genomic DNA preps: Has anyone had success preparing genomic DNA for Southerns using the DNeasy kit from Qiagen?
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• Genomic DNA preps: I was wondering if anyone has advice on using BRL/Life's Trizol and Dnazol to isolate RNA/ genomic DNA from Dictyostelium?
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Cloning and PCR Amplification

• Gel purification of DNA fragments
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• I was wondering whether anyone has handy a protocol for isolating extrachromosomal plasmids from Dictyostelium cells.
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• Cloning upstream gene sequences: I am interested to know if anyone has experience cloning unknown upstream Dicty genomic DNA using PCR based on a known cDNA sequence?
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• Stability of Dicty DNA in bacteria: Does anyone have magical tricks to deal with these problems?
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• Would anybody know of a high-fidelity polymerase that could PCR-amplify very AT-rich regions?
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RNA extraction and Nothern blots

• Problems with RNA extraction in AX4: Does anybody know that if there is any difference between AX2 and AX4?
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• RNA extraction: I need some more details on extracting RNA from synchronized developing Dicty on filters.
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• Does anyone have a preferred method for isolating polyA-RNA from vegetative cells?
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• What is the best gene for standardizing Northern blot during Dicty vegetative stage?
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• What is the best gene for standardize Northern blot during dicty development?
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• What can be the internal control for RT-PCR to investigate mRNA expression level during dicty development?
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• β-gal Northerns: I´m using β-gal as a reporter gene in Dicty and I get nice protein expression. Unfortunately I can hardly detect the mRNA in Northern blots.
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Probes

• Does anyone have a favorite non-isotopic method/kit for making probes?
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• I am looking for an 18s primer for Dictyosteloids.

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Biochemistry Techniques

• How to break cells without detergent?
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• We are in the search of a good secondary antibody to detect anti-mouse antibodies in Western blot.
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• Is there a consensus of protease inhibitor combinations to work with Dicty cell lysates?
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• Does anyone have any experience in making really clean immunopreciptations from Dicty?
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• I'm trying to optimize a rapid centrifugation protocol to pellet Dicty. The goal here is to efficiently pellet Dictyostelium whole cells in suspension, as rapidly as possible, without disruption of the plasma membrane.
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• Has anyone used the Ras activation kit (from any company) to do the Ras activation assay in Dictyostelium cells?
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• I am planning to purchase a PKA assay kit to measure PKA activity with PKA inhibitor treatment over time. Anybody have a suggestion for me about what I should get?
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• Metabolic labelling of cells: Does anyone have a good protocol for metabolic labelling of proteins with 35S? I have been told that Dicty do not take up 35S-methionine particularly well. Does anyone know if this is true or false?
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• Has anyone done an 35-S-amino acid pulse-chase experiment recently or know of a good reasonably recent reference where it worked?

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Dictyostelium Proteins

• Has anyone successfully demonstrated release of GPI anchored proteins with phospholipase C on cells or membranes?
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• Does anyone have an idea of the frequency of N-terminal blocking in Dicty proteins?
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• Has anyone observed more than nine mannose residues on N-linked glycostructures on D. discoideum expressed proteins?
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• Has anybody ever shown experimentally (e.g. by site-directed mutagenesis) what nuclear targeting signals (NLS) might look like in Dictyostelium?
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• Protein identification: Does anyone know of a reputable facility in which mass spec and/or protein sequencing can be performed on the individual bands?
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• What are the major proteins in a growing Dictyostelium cell? For "major" I mean protein visible on a Coomassie stained SDS page minigel.

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Cytoskeleton

• Isolation of actin-binding proteins: Does anybody have experience on depolymerization of cortex? I try to isolate a actin binding protein from cortex prepared from Triton-treated Dicty cells.
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• Does anyone know of any methods to label, exclusive of one another, either the F or G actin in a cell in a way that leaves the cell alive?
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• Microtubule staining: We are trying to stain microtubular cytoskeleton in Dictyostelium amoebas by means of immunocytochemistry technique. We have tried several protocols described in the literature but they failed to give acceptable results.
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• We want to disrupt the microtubule network in Dicty.
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• I am looking for a good protocol for performing a Triton Insoluble Cytoskeleton Test.
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• I can't find any reference to intermediate filaments in Dictyostelium. Do they exist? Are some genes identified?

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Cell Biology Methods

• I would like to block exocytosis in Dicty without affecting morphogenesis.
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• Does anyone know how to extract intact lysosomes?
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• Is there a generally-accepted method for quantifying Dicty cell growth rates when the cells are grown ON A SURFACE?
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• Does anyone know of a good method for permeabilizing Dicty?
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• Experiences with Lysotracker.
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• Does anyone have experience/knowledge about possibility of pulling micropipettes for chemotaxis experiments with vertical pipette pullers?
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• I would appreciate any assistance on the folate chemotaxis method using a micropipet.
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• FAB - Cell adhesion and phagocytosis assay protocols
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• I would like to determine the rates at which different strains of Pseudomonas aeruginosa are taken up by Dictyostelium.
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• I am trying to label bacteria with Texas Red. Pseudomonas aeruginosa stains nice and bright, but Klebsiella aerogenes stains rather poorly.
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• We are interested in phagocytosis by the D. discoideum amoeba, and classicaly use fluorescent bacteria to assay phagocytosis. What is known about feeding behavior of Dictyo, can they eat other microorganisms, such as paramecium or others? Are there bacteria or others that Dictyo CANNOT kill?
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• Can anybody please give me some advice on the synchronisation of Dictyostelium?
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• Looking to buy a micromanipulator

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• Microscopy

• Does anyone know how to fix dicty cells for immunofluorescence studies?
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• Can someone direct me to a method for disaggregating cells from mounds and slugs that yields cells suitable for immunofluorescent microscopy?
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• Autofluorescence: I am visualising live cells grown in HL5, and have a problem with autofluorescence.
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• Autofluorescence: Purging background green fluorescence.
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• Cell fixation: I would like to find a protocol to fix Dicty cells expressing GFP such that GFP fluorescence is maintained and nuclei can be stained with propidium iodide.
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• Phototoxicity: Has anyone done a study in any depth about the worst and best wavelengths for photoxicity in Dicty?
  
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• Digital Cameras: I am interested in buying a digital camera for our Nikon dissecting scope. Experience here is leading us to the KODAK DCS (either 420 or 460). I am looking for any advice others may have with these or alternative configurations.

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Cell Staining

• Does any one know a cell viability assay that works best for dicty?
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• Hoechst 33342/33258: Does anyone know a procedure for DNA staining in living Dicty cells?
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• Is the Molecular probe TOTO dye able to stain LIVE gram negative bacteria or is there any other RED (or any color other than green) fluorescent dyes capable of doing the job???
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• We would like to be able to visibly stain amoebae adhered to plastic dishes to aid in the quantitation and imaging of clonal foci such as those obtained by transformation.

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Drugs, Inhibitors, Reagents

Reagents

• Can anyone suggest a type of agarose to use that is comparable to agarose-M from LKB Pharmacia?
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• Is DIF available for purchase from specific companies?
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Antibodies

• Does anyone know which company's anti-GFP antibodies worked well in co-IP experiment?
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• Does anyone know of a good anti-tubulin antibody that works well in Dicty?
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• I am trying to do immunofluorescence studies with an anti-tubulin antibody in order to visualize microtubules. I was wondering which anti-tubulin antibody would be recommended, and what company I might be able to purchase it from.
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• I'm looking for antibodies against Dicty organelle markers (ER, Golgi, endosomes,...)
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• I'm looking for a commercially available anti myc-antibody that works well with myc-tagged proteins in Dicty (primarily for westerns but maybe also for indirect immunofluorescence)
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Drugs and Inhibitors

• Does anyone know if aluminum fluoride or GTPγs were successfully used to mimic activation of Gα in living Dicty?
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• May anyone suggest me dose and time effect for Actinomycin D in Dd?
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• Inhibition of RNA synthesis
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• I want to block protein synthesis in Dicty without affecting endocytosis and phagocytosis.
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• Has anybody used proteosome inhibitors in Dicty?
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