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Bodipy fl c5 ceramide

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BODIPY FL C5 ceramide is a fluorescent lipid analogue used to label cellular membranes and study their dynamics. It consists of a BODIPY fluorescent dye attached to a five-carbon fatty acid chain, which allows it to incorporate into cellular lipid bilayers.

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Lab products found in correlation

Lsm 710 confocal microscope, by Zeiss (3 mentions) Lysosensor green, by Thermo Fisher Scientific (2 mentions) Organelle trackers, by Thermo Fisher Scientific (2 mentions) Er tracker blue white, by Thermo Fisher Scientific (2 mentions) Mitotracker green, by Thermo Fisher Scientific (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) O phenanthroline, by Merck Group (1 mentions) Pepstatin a, by Enzo Life Sciences (1 mentions) Diic18, by Thermo Fisher Scientific (1 mentions) A 350, by Thermo Fisher Scientific (1 mentions) C12 cer, by Avanti Polar Lipids (1 mentions) Piperacillin, by Merck Group (1 mentions) Mecillinam, by Merck Group (1 mentions) D cycloserine, by Merck Group (1 mentions) Affinity purified mouse monoclonal antibodies, by BioLegend (1 mentions) Penicillin g, by Merck Group (1 mentions) Leica dm6b upright microscope, by Leica camera (1 mentions) Axiovert 200m microscope, by Zeiss (1 mentions) Glass bottom dishes, by MatTek (1 mentions) Lipidtox deep red, by Thermo Fisher Scientific (1 mentions)

16 protocols using bodipy fl c5 ceramide

1

BODIPY FL C5-Cer Trafficking Assay

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For BODIPY FL C5‐Cer (BODIPY™ FL C5‐Ceramide complexed to BSA, ThermoFisher) transport to the Golgi and MVE/LE, HeLa WT or CERT‐deficient cells were plated onto bottom glass six wells plates with 14 mm microwells (Cellvis P06‐12‐0‐N). For the Golgi transport, on the day of the experiment cells, were pre‐treated with PI4KIIIβ‐IN‐10 (25 nM and 50 nM) and NC03 (5 and 10 μM) for 15 min. Next, cells were incubated on ice for 20 min with 0. 5 μM BODIPY FL C5‐Cer in phenol red and serum‐free DMEM, and the excess fluorescent dye was removed by washing with serum‐free DMEM. Next, BODIPY FL C5‐Cer was allowed to be distributed in cells for 10–15 min at 37°C before cells were fixed in 2.5% PFA and 2.5% glutaraldehyde. For the MVE/LE transport, HeLa WT or CERT‐deficient cells were transfected with a fusion construct of Rab7a and TagRFP (CellLight™ Late Endosomes‐RFP, BacMam 2.0, ThermoFisher) 24 h before incubation with BODIPY FL C5‐Cer. The distribution of BODIPY FL C5‐Cer was analyzed with the Eclipse Ti2‐E inverted confocal microscope using a 100x objective. A minimum of four images with 9–13 planes in Z‐direction were acquired for each condition and deconvolved before quantifying Golgi mean fluorescent intensities (MFI) or BODIPY FL C5‐Cer correlation to late endosomes‐RFP. MFI and correlation were assessed with the Nikon analyzer software.
Crivelli S.M., Giovagnoni C., Zhu Z., Tripathi P., Elsherbini A., Quadri Z., Pu J., Zhang L., Ferko B., Berkes D., Spassieva S.D., Martinez‐Martinez P, & Bieberich E. (2022). Function of ceramide transfer protein for biogenesis and sphingolipid composition of extracellular vesicles. Journal of Extracellular Vesicles, 11(6), e12233.
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2

Visualizing Chlamydia Lipid Trafficking

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Cells were seeded in glass bottom dishes and infected with C. trachomatis at an MOI of 0.5. Five µM BODIPY FL-C5 Ceramide (Thermo Fisher Scientific) was added in the culture medium at 23 hpi for 30 min at 4°C. After removing ceramide from the culture medium, cells were incubated at 37°C up to 24 and 36 hpi.
Suzuki M., Funakoshi T., Kumagai K., Komatsu M, & Waguri S. (2023). ATG9A supports Chlamydia trachomatis infection via autophagy-independent mechanisms. Microbiology Spectrum, 11(5), e02774-23.
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3

Lipid Biosynthesis and Membrane Dynamics

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The following reagents were used: THC (THC Pharm GmbH, THC-1099), pepstatin A (Enzo Life Sciences, ALX-260-085), E64d (Enzo Life Sciences, BML-PI107), myriocin (ISP-1, Sigma-Aldrich, M1177), sphingomyelinase (EC 3.1.4.12) from Bacillus cereus (Sigma-Aldrich, S7651), o-phenanthroline (Sigma-Aldrich, 131377) and CA-074 methyl ester (CTSB/cathepsin B inhibitor; Sigma-Aldrich, C5857). Phosphatidylcholine from egg yolk (PC; Lipid Products, grade 1, 840051P), sphingomyelin, (SM; Avanti Polar Lipids, 860061), C12 ceramide (C12-Cer; Avanti Polar Lipids, 860512), C16 dihydroceramide (C16-dhCer; Avanti Polar Lipids, 860634), palmitoyl-oleoylphosphatidylcholine (POPC; Avanti Polar Lipids, 850457) and cholesterol (Ch; Avanti Polar Lipids, 700000). DEGS1 inhibitor GT11, and dihydrosphingomyelin (dhSM) were synthesized in our laboratories. dhSM was synthesized from egg SM (Avanti Polar Lipids, 860061) and contained 86% C16 dhSM. ANTS (Molecular Probes, Inc., A350), DPX (Molecular Probes, Inc., X1525) and DiIC18 (Molecular Probes, D3911). BODIPY FL C5-ceramide complexed to BSA (ThermoFisher Scientific, B22650).
Hernández-Tiedra S., Fabriàs G., Dávila D., Salanueva Í.J., Casas J., Montes L.R., Antón Z., García-Taboada E., Salazar-Roa M., Lorente M., Nylandsted J., Armstrong J., López-Valero I., McKee C.S., Serrano-Puebla A., García-López R., González-Martínez J., Abad J.L., Hanada K., Boya P., Goñi F., Guzmán M., Lovat P., Jäättelä M., Alonso A, & Velasco G. (2016). Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization. Autophagy, 12(11), 2213-2229.
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4

Chlamydia MOMP Immunodetection Protocol

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Goat polyclonal antibodies directed against the Chlamydia major outer membrane protein (MOMP) were purchased from Meridian Life Science. Click-It Labeling Kit for the detection of EDA-DA and various Alexa Fluor conjugated secondary antibodies were purchased from Invitrogen. Affinity purified mouse monoclonal antibodies directed against the E. coli RNA polymerase β subunit, which crossreact with the chlamydial RNA polymerase β subunit, were purchased from Biolegend. BODIPY FL C5 ceramide was purchased from Invitrogen. Hoechst 33342 was purchased from ThermoFisher. Penicillin G, piperacillin, d-cycloserine, and mecillinam were purchased from Sigma Chemicals. A22 was purchased from Cayman Chemical.
Cox J.V., Abdelrahman Y.M, & Ouellette S.P. (2020). Penicillin-binding proteins regulate multiple steps in the polarized cell division process of Chlamydia. Scientific Reports, 10, 12588.
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5

Organelle Visualization in HEp2 Cells

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The human HEp2 cells were incubated in a 35 mm tissue culture dish (CELLTREAT) and allowed to grow overnight. The cells were exposed to each compound at a concentration of 10 μM and incubated for 6 h (5% CO2, 37 °C). The following organelle trackers (Invitrogen) were added to the cells: ER Tracker Blue/White (100 nM), BODIPY FL C5 Ceramide (50 nM), MitoTracker Green (250 nM), LysoSensor Green (50 nM). The cells were incubated with each compound and trackers for half an hour, and washed with PBS solution three times before imaging. A Leica DM6B upright microscope equipped with a water immersion objective, and DAPI, GFP, and Texas Red filter cubes (Chroma Technologies) were used to acquire the images.
Zhao N., Williams T.M., Zhou Z., Fronczek F.R., Sibrian-Vazquez M., Jois S.D, & Vicente M.G. (2017). Synthesis of BODIPY-Peptide Conjugates for Fluorescence Labeling of EGFR over-expressing Cells. Bioconjugate chemistry, 28(5), 1566-1579.
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6

Time-lapse Imaging of Chlamydia Ceramide

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HeLa cells were seeded in glass bottom dishes (MatTek, Ashland, MA) and infected with C. trachomatis L2, with or without addition of 100 U/ml PenG, for 24 h. The medium was replaced with fresh medium containing 100 nM BODIPY FL C5 ceramide (Invitrogen #B22650). After 30 minutes, cells were subjected to time lapse microscopy at 37°C in a chamber with humidified atmosphere (6.5% CO2 and 9% O2). An inverted Axiovert 200 M microscope (Zeiss) equipped with a Yokogawa CSU-X1 spinning disc confocal head (Tokyo, Japan), an emission filter wheel, a Coolsnap HQ II digital Camera (Roper Scientific, Tucson, AZ) and driven by Metamorph imaging software version 7.7.11.0 (Universal Imaging) was used to collect images. BODIPY FL C5 ceramide was excited with a 488 nm solid state laser. Fluorescence intensities were measured inside the inclusion in a 34 µm2 area (acute n = 35; persistent n = 26 inclusions) at the indicated time points. Averages and SEM of two independent experiments were calculated using Microsoft Excel 2010.
Dille S., Herbst K., Volceanov L., Nölke T., Kretz O, & Häcker G. (2014). Golgi Fragmentation and Sphingomyelin Transport to Chlamydia trachomatis during Penicillin-Induced Persistence Do Not Depend on the Cytosolic Presence of the Chlamydial Protease CPAF. PLoS ONE, 9(7), e103220.
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7

Visualizing Ceramide Trafficking and RhoA Dynamics

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AlexaFluor546 (Alexa546), LipidTOX Green, LipidTOX Deep Red, Alexa488-phalloidin, Alexa647-phalloidin, and BODIPY-FL-C5-ceramide were purchased from Invitrogen. C16-ceramide (d18:1/16:0) and C2-ceramide (d18:1/2:0) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Monoclonal anti-ceramide antibody (clone MID 15B4) and desipramine were purchased from Sigma-Aldrich (St. Louis, MO). Y-27632 was purchased from Tocris Bioscience (Bristol, UK). Rho inhibitor I was purchased from Cytoskeleton, Inc. (Denver, CO). Phospho(Thr18/Ser19)-Myosin Light Chain 2 antibody was purchased from Cell Signaling Technology (Danvers, MA). Anti-RhoA and anti-calnexin antibodies were purchased from Abcam (Cambridge, MA). pcDNA3-enhanced green fluorescent protein (EGFP) was a gift from Doug Golenbock (Addene plasmid #13031). pcDNA3-EGFP-RhoAwt [WTRhoA-green fluorescent protein (GFP)] and pcDNA3-EGFP-RhoAQ63L (CARhoA-GFP) were a gift from Gary Bokoch (Addgene plasmid #12965 and #12968).
Singh R.K., Haka A.S., Brumfield A., Grosheva I., Bhardwaj P., Chin H.F., Xiong Y., Hla T, & Maxfield F.R. (2017). Ceramide activation of RhoA/Rho kinase impairs actin polymerization during aggregated LDL catabolism. Journal of Lipid Research, 58(10), 1977-1987.
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8

Visualizing AV Canal Valve Formation in Zebrafish

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Bodipy FL C5-Ceramide (Invitrogen D3521) was added to fish media 1 day prior to imaging at 0.2 µM from a 1 mM stock solution. The larvae were anesthetized in 0.0175% tricaine and their heart stopped using 25 mM BDM (Sigma Aldrich B0753) treatment. Images were then acquired using an LSM 700 confocal laser scanning microscope (Zeiss) using a 40x objective, and the presence of a superior valve leaflet was assessed in every plane of a z-stack covering the AV canal. The 78 hpf wild-type dataset (n = 104) is the same in Figs. 1k, 2l, 3j, 6o, and represents a pool of pkd1a+/+, pkd1a+/+; pkd2+/+, and camk2g1+/+; camk2g2+/+ larvae. The 78 hpf pkd1a mutant dataset (n = 32) is the same in Figs. 1k, 2l, 3j.
Juan T., Ribeiro da Silva A., Cardoso B., Lim S., Charteau V, & Stainier D.Y. (2023). Multiple pkd and piezo gene family members are required for atrioventricular valve formation. Nature Communications, 14, 214.
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9

Cuticle Reduction and Staining Protocol

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Animal fixation and cuticle reduction were adapted from40 (link). Worms were fixed with pre-chilled 4% paraformaldehyde in PBS at room temperature on a rotator for 30 min and moved to 4 °C overnight. Animals were then washed three times with reduced cuticle buffer before incubating with fresh reduced cuticle buffer at 4 °C for 24 h. Afterwards, reduced animals were treated with 0.5 kU/mL collagenase type VII (Sigma–Aldrich, C0773) at 37 °C for ~32 h. Afterwards, animals were rinsed once with DI water, followed by 3 washes with PBS, and store in PBS at 4 °C for 1 day before staining. Animals were stained first with 0.5 mg/mL DAPI overnight. Excess DAPI was removed before staining with respective dyes. For membranes with a mixture of CellBrite Cytoplasmic membrane dye (Biotum, 30021) and BODIPY FL C5-Ceramide (Invitrogen, B22650) at 400- and 1000-fold dilution, respectively, for 2 days. For phalloidin staining, Alexa 488-tagged phalloidin (Invitrogen, 400× stock, A12379) was added at 4× working concentration and incubated for 2 days. All the staining was carried out in a thermal shaker at 37 °C, 900 rpm.
Tran T.D., Ali M.A., Lee D., Félix M.A, & Luallen R.J. (2022). Bacterial filamentation as a mechanism for cell-to-cell spread within an animal host. Nature Communications, 13, 693.
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10

Fluorescent Organelle Tracking in HEp2 Cells

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The HEp2 cells were placed in 6 well plates (celltreat.com), allowed to grow overnight, and then exposed to 10 μM concentration of the compound for 6 h. After exposure to the compound, the following organelle trackers (Invitrogen) and amounts were added to the cells: ER Tracker Blue/White (100 nM), BODIPY FL C5 Ceramide (50 nM), MitoTracker Green (250 nM), LysoSensor Green (50 nM), for 30 min in a 37 oC incubator. The cells were washed with 1X PBS three times, and then refilled with 1X PBS 5 mL/well. The distribution of the compounds was determined using an upright Leica DM6B fluorescence microscope fitted with standard Texas Red, GFP, DAPI, filter sets. A water immersed objective was used to determine the compound localizations.
Williams T.M., Zhou Z., Singh S., Sibrian-Vazquez M., Jois S.D, & Vicente M.G. (2020). Targeting EGFR Overexpression at the Surface of Colorectal Cancer Cells by Exploiting Amidated BODIPY-Peptide Conjugates. Photochemistry and photobiology, 96(3), 581-595.
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