The axenic Dictyostelium discoideum wild-type strain was used for all experiments. Cells were grown in HL5-C medium (Formedium) at 22 °C. The indicated constructs were transformed in AX2 cells by electroporation and selected with either 10 µg/mL geneticin or 50 µg/mL hygromycin B. To induce expression from the tetracycline inducible vectors, the cells were overnight incubated with 1 µg/mL doxycycline. Dimerization of FKBP12-GFP was induced by incubation with B/B homodimerizer (Clontech, also called AP20187) to a final concentration of 1 µM for 3 h.
Hl5c medium
Manufactured by Formedium
Sourced in United Kingdom
The HL5c medium is a nutrient-rich culture medium used for the growth and maintenance of various cell lines in laboratory settings. It provides the necessary components to support the proliferation and viability of cells under controlled conditions.
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Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
B b homodimerizer, by Takara Bio (1 mentions)
Tcs sp2 confocal laser scanning microscope, by Leica (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Pgem t easy, by Promega (1 mentions)
Tween 80, by Merck Group (1 mentions)
Fluorescein diacetate, by Merck Group (1 mentions)
Mg oac 2, by Merck Group (1 mentions)
Nh4oac, by Merck Group (1 mentions)
Kh2po4, by Merck Group (1 mentions)
Nabh4, by Merck Group (1 mentions)
Na2hpo4, by Merck Group (1 mentions)
Agno3, by Merck Group (1 mentions)
Geneticin, by Merck Group (1 mentions)
N propyl gallate, by Merck Group (1 mentions)
N n n n tetramethyl ethylenediamine tmeda, by Merck Group (1 mentions)
24 well cell culture plates, by Sarstedt (1 mentions)
α bromoisobutyryl bromide, by Merck Group (1 mentions)
15 protocols using hl5c medium
1
Inducible Dictyostelium Dimerization Assay
2
Localization of Dictyostelium DyPA Protein
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Dictyostelium DyPA expression constructs with N- and C-terminal EYFP fusions were generated in the plasmids pDXAYFPmcs and pDXAmcsYFP, respectively [69 (link)]. The DNA fragment encoding DyPA cDNA was inserted between BamHI and XhoI sites by conventional PCR using Dictyostelium gDNA. All the constructs were verified through DNA sequencing.
Dictyostelium discoideum AX2 cells were grown in HL-5C medium (Formedium) at 21°C. Cells were transformed with the expression constructs by electroporation as described previously [70 (link),71 (link)]. Transformants were selected in the presence of 10 µg/mL G-418 (Formedium). Dictyostelium discoideum AX2 cells were grown on glass-bottom petri plates (MatTek Corp) to 50–60% confluency for confocal microscopy imaging. Imaging was performed in a buffer containing 10 mM MES-NaOH pH 6.5, 2 mM MgCl2, 0.2 mM CaCl2, at 512 nm with a Leica TCS SP2 confocal laser scanning microscope equipped with a 63 × 1.4 NA HCX PL APO CS oil immersion objective. Experiments were performed at room temperature.
Dictyostelium discoideum AX2 cells were grown in HL-5C medium (Formedium) at 21°C. Cells were transformed with the expression constructs by electroporation as described previously [70 (link),71 (link)]. Transformants were selected in the presence of 10 µg/mL G-418 (Formedium). Dictyostelium discoideum AX2 cells were grown on glass-bottom petri plates (MatTek Corp) to 50–60% confluency for confocal microscopy imaging. Imaging was performed in a buffer containing 10 mM MES-NaOH pH 6.5, 2 mM MgCl2, 0.2 mM CaCl2, at 512 nm with a Leica TCS SP2 confocal laser scanning microscope equipped with a 63 × 1.4 NA HCX PL APO CS oil immersion objective. Experiments were performed at room temperature.
3
Visualizing Actin and Microtubules in Dictyostelium
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Dictyostelium discoideum cells expressing the green fluorescent GFP-LimΔcoil fusion protein [25 (link)] were cultivated on mineralized and unmineralized wafers at 21 °C in HL5c medium (Formedium, Hunsanton, UK). All samples were incubated for 60 h; then the adhering cells were fixed with glutaraldehyde (0.5%) [26 ]. Actin appears green as GFP-Limcoil fusion protein binds to F-actin. Microtubules were visualized in red using the monoclonal anti-α-tubulin antibody YL1/2 and the anti-rat-antibody AlexaFluor-568. Cell nuclei were labeled in blue with 4′,6-diamidino-2-phenylindol dihydrochloride (DAPI). N-propylgallate (2%) was used as anti-bleaching agent and samples were mounted in Mowiol [26 ]. Wide-field microscopy was done on a Zeiss CellObserver HS/Axiovert 200M system with a PlanApo 100×/1.4 N.A. Lens and an Axiocam MRm Rev. 3 CCD Camera. z-Stacks were recorded at a distance of 0.25 µm. Iterative deconvolution of microscopic images with a measured point spread function was performed with Zeiss Axiovision 4.8. Data analysis was carried out with ImageJ 1.48k (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/ , 1997–2014.)
4
Dictyostelium Centrosome Isolation and BioID Assays
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Cells were cultured in HL5c medium (Formedium, Hunsanton, UK). Sterile filtered glucose was added after autoclaving. Transformation of Dictyostelium amoebae by electroporation, SDS electrophoresis and Western blotting were carried out according to standard procedures [25 (link),29 (link)]. Centrosomes or nuclei with attached centrosomes and were isolated as reported earlier [30 (link)]. BioID assays were performed as described recently [20 (link),30 (link)].
5
Culturing Dictyostelium for M. marinum Infection
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D. discoideum AX2–214 (DBS0235534, www.dictybase.org ) was used for all experiments in connection with M. marinum infection and was kindly provided by Thierry Soldati. D. discoideum cells were cultured axenically at 22 °C in 10 ml HL5-C medium (pH 6.4) (Formedium) supplemented after autoclaving with 1% glucose and 100 U/ml Penicillin-Streptomycin (Gibco by Life Technologies). The cells were grown in BD Falcon tissue culture dishes (100 × 20 mm). Antibiotics were excluded from the growth medium the day before and during infection experiments.
6
Genetically Engineered Dictyostelium Strains
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The D. discoideum strains used for this work are listed in S1 Table . Cells were axenically grown at 22°C in HL5c medium (Formedium) supplemented with 100 U mL−1 of penicillin and 100 μg mL−1 of streptomycin (Invitrogen).
To generate the p62 knockout strain, p62 was amplified with the primers p62F10 and p62R10 from genomic DNA of DH1 and ligated into pGEM-T-Easy (Promega). The Bs-resistance cassette flanked by SmaI sites from plasmid pLPBLP [61 (link)] was inserted into pGEM-T-p62, previously digested with EcoRV and BclI and blunted.
Plasmids and primers to generate the D. discoideum GFP-tagged proteins Atg8, Atg18, Atg1, Ub, p62, Rab11c, Rab7a, VatB, Lamtor1, Rheb, Lst8 and Raptor are listed inS1 and S2 Tables. Plasmids were transfected into D. discoideum by electroporation and selected with the relevant antibiotic. Hygromycin and G418 were used at a concentration of 50 and 5 μg mL−1, respectively.
To generate the p62 knockout strain, p62 was amplified with the primers p62F10 and p62R10 from genomic DNA of DH1 and ligated into pGEM-T-Easy (Promega). The Bs-resistance cassette flanked by SmaI sites from plasmid pLPBLP [61 (link)] was inserted into pGEM-T-p62, previously digested with EcoRV and BclI and blunted.
Plasmids and primers to generate the D. discoideum GFP-tagged proteins Atg8, Atg18, Atg1, Ub, p62, Rab11c, Rab7a, VatB, Lamtor1, Rheb, Lst8 and Raptor are listed in
7
Dictyostelium Cell Culture and Protein Analysis
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Dictyostelium cells (strain AX2) were cultured in HL5c medium (Formedium, Hunsanton, UK) at 21 °C supplemented with sterile-filtered glucose added after autoclaving, either adherently in tissue culture flasks for transformation by electroporation [34 (link)], or in suspension in Erlenmeyer flasks on a rotary shaker at 150 rpm for protein expression. SDS electrophoresis and Western blotting with either nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate (NBT/BCIP) color detection or enhanced chemiluminescence was performed as described earlier [23 (link)].
8
Culturing Bacterial and Eukaryotic Cells
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Bacterial strains used in this study are listed in Table 1 . M. marinum M strain was grown in Middlebrook 7H9 (Difco) supplemented with 10% OADC (Becton Dickinson), 0.2% glycerol (Biosciences), and 0.05% Tween 80 (Sigma) at 32°C in shaking culture at 150 rpm in the presence of 5 mm glass beads to prevent aggregation. The axenic D. discoideum Ax2(Ka) was cultured at 22°C in Hl5c medium (Formedium) supplemented with 100 U/mL of penicillin and 100 µg/mL of streptomycin (Invitrogen). BV2 cells were grown at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 6% of heated inactivated fetal bovine serum (FBS) and 100 U/mL of penicillin in a 5% CO2 environment.
9
DNA Structural Characterization
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DNA strands 5′-CAC CGC TTT TGC CTT TTG GGG ACG GAT A-3′ (28b), 5′-TGC CTT TTG GGG ACG GAT A-3′ (19b), and (b) 5′-CT TAC CTC CCC CCC CCC CCA GGT AAG-3′ (HP) with standard desalting (Integrated DNA Technologies, USA), AgNO3 (99.9999%, Sigma Aldrich, The Netherlands), NH4OAc (99.99%, Sigma Aldrich, The Netherlands) NaBH4 (99%, Sigma Aldrich, The Netherlands), KH2PO4 (>99%, Sigma Aldrich, The Netherlands), Na2HP, O4 (>99%, Sigma Aldrich, The Netherlands), Maltose (Duchefa Biochemie B. V., Netherlands), Fluorescein diacetate (F7378, Sigma Aldrich Merck, Germany), HL5-C medium (Formedium™, United Kingdom), NaCl (Fisher Scientific Company LLC, USA), Mg(OAc)2 (>99%, Sigma Aldrich, The Netherlands), Geneticin (Sigma Aldrich, The Netherlands), 3 kDa and 50 kDa centrifugal filter (Merck Millipore, Germany) were used as received without further purification. Bi-distilled water from a Millipore system (Milli-Q Academic A-10, Merck Millipore Germany) was used for all solutions.
10
Antibiotic Selection of Adherent Cells
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HL5c medium (Formedium, Hunstanton, UK) supplemented with sterile filtered glucose was used. Clones were selected with 10 µg/mL G418 or 4 µg/mL Blasticidin S or 50 µg/mL hygromycin, respectively. Cells were grown in adherent culture using tissue culture flasks.
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