GPMVs were prepared through incubation with low concentrations of dithiothreitol (DTT, 2mM) and formaldehyde (25mM) in the presence of calcium (2mM) for 1h as described previously [1 (link),6 (link),10 (link),23 (link)]. Prior to GPMV formation, cells were labeled with DiI-C12 (Life Technologies, Carlsbad, CA; 2μg/ml in 1% methanol) for 10min. GPMVs were imaged on an inverted microscope (IX81; Olympus, Center Valley, PA) with a 40x air objective (0.95 NA), epi-illumination using an Hg lamp and Cy3 filter set (Chroma Technology, Bellows Falls, VT). Temperature was controlled using a home built peltier stage described previously [23 (link),24 (link)] coupled to a PID controller (Oven Industries, Mechanicsburg, PA), and images were recorded using a SCMOS camera (Neo; Andor, South Windsor, CT).
Dii c12
Manufactured by Thermo Fisher Scientific
Sourced in United States
DiI-C12 is a lipophilic dye used for cellular labeling and membrane staining. It is a long-chain carbocyanine dye that incorporates into the lipid bilayer of cell membranes.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (2 mentions)
Cacl2, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Biotin azide, by Cayman Chemical (2 mentions)
Anti myc alexafluora 647, by Cell Signaling Technology (2 mentions)
Tris 2 carboxyethyl phosphine tcep, by Research Products International (2 mentions)
Tris 1 benzyl 1h 1 2 3 triazol 4 yl methyl amine tbta, by Thermo Fisher Scientific (2 mentions)
Anti myc magnetic beads, by Thermo Fisher Scientific (2 mentions)
Anti myc, by Cell Signaling Technology (2 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes, by Research Products International (2 mentions)
Cucl2, by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Research Products International (2 mentions)
Hela cell line, by American Type Culture Collection (2 mentions)
Goat antihuman, by LI COR (2 mentions)
Goat anti mouse igg, by LI COR (2 mentions)
Mtz nhs, by Vector Laboratories (2 mentions)
Dii c18 2, by Thermo Fisher Scientific (2 mentions)
Diic16, by Thermo Fisher Scientific (2 mentions)
Dii c18, by Biotium (2 mentions)
8 protocols using dii c12
1
Generating Plasma Membrane Vesicles for Biophysical Analysis
2
Biochemical Pulldown Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DiIC12 was purchased from Life Technology. NBD-PE was purchased from Avanti. anti-myc AlexaFluora-647, anti-biotin, and anti-myc were purchased from Cell Signaling Technologies. anti-myc AlexaFluora-647 labeled antigen binding fragment (Fab) was purchased from Promega. DTT, Tris(2-carboxyethyl)phosphine (TCEP), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) were purchased from Research Products International; DTT was prepared fresh for every use. Paraformaldehyde (PFA) was purchased as a 16% stock solution from Electron Microscopy Sciences. CaCl2, NaCl, CuCl2, Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), anti-myc magnetic beads, and propidium iodide were purchased from Fisher Scientific. 17-ODYA and biotin-azide were purchased from Cayman Chemicals. HeLa cell lines were acquired from American Type Culture Collection. Primary RSCs were a generous gift from the laboratory of Bruce Carter at Vanderbilt University (Nashville, TN).
3
Cell Membrane Protein Labeling and Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DiIC12 was purchased from Life Technology (Eugene, USA). NBD-PE was purchased from Avanti (Alabaster, USA). anti-myc AlexaFluora-647, anti-biotin, and anti-myc were purchased from Cell Signaling Technologies (Danvers, USA). DTT, tris(2-carboxyethyl)phosphine (TCEP), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Research Products International (Mount Prospect, USA); DTT was prepared fresh for every use. Paraformaldehyde (PFA) was purchased as a 16% stock solution from Electron Microscopy Sciences (Hatfield, USA). CaCl 2 , NaCl, CuCl 2 , Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), anti-myc magnetic beads and propidium iodide were purchased from Fisher Scientific (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted January 29, 2020. ; https://doi.org/10.1101/2020.01.28.923771 doi: bioRxiv preprint (Fair Lawn, USA). 17-Octadecynoic Acid (17-ODYA) and biotin-azide were purchased from Cayman Chemicals (Ann Arbor, USA). HeLa cell lines were acquired from ATCC (Manassas, USA). Primary rat Schwann cells (RSCs) were a generous gift from the lab of Dr. Bruce Carter at Vanderbilt University.
The copyright holder for this preprint this version posted January 29, 2020. ; https://doi.org/10.1101/2020.01.28.923771 doi: bioRxiv preprint (Fair Lawn, USA). 17-Octadecynoic Acid (17-ODYA) and biotin-azide were purchased from Cayman Chemicals (Ann Arbor, USA). HeLa cell lines were acquired from ATCC (Manassas, USA). Primary rat Schwann cells (RSCs) were a generous gift from the lab of Dr. Bruce Carter at Vanderbilt University.
4
Membrane Fluidity Visualization with DiIC12
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect regions of increased membrane fluidity, staining with DiIC12 (Thermo Fisher) staining was performed. Bamy strains were grown overnight on King’s B plates and then resuspended in 1 mL dH2O followed by addition of 1 μg ml−1 DiIC12. 0.5% Benzyl-alcohol was added to positive control samples. Cells were incubated at 28°C for 3 hours and then washed four times and resuspended in dH2O. Images were obtained by visualizing samples using a Leica SP5 confocal microscope with a 63x NA 1.3 Plan APO oil immersion objective and acquisitions with excitation at 651 nm. Image processing was performed using FIJI/ImageJ software. For each experiment, laser settings, scan speed, PMT detector gain, and pinhole aperture were kept constant for all acquired image stacks.
5
Lipid Labeling for Cellular Imaging
6
Colon Cancer Cell Transfection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glutamine, DMEM, trypsin-EDTA, and penicillin/streptomycin were obtained from GE Healthcare HyClone (Logan, UT, United States). Fetal bovine serum, enzymes, 3-aminopropyltriethoxy silane (TESPA), L-15 and chemicals were from Sigma (St. Louis, MO, United States). DiI-C12, DiD-C18, Lipofectamine 2000 and Gibco Opti-MEM were from Thermo Fisher Scientific (Waltham, MA, United States). High performance, 1.5H, coverslips were from Marienfeld (Lauda-Königshofen, Germany). The colon cancer cell line HT29 was a kind gift of Dr. A. Blokzijl, Uppsala University, Sweden and SW480 cells were from ATCC (Manassas, VA, United States). CD3ζ-EYFP, mouse CD3ζ fused to EYFP via a six aa linker expressed in the pBJ1-Neo plasmid under the CMV-promotor, was from Mark Davis, Stanford University School of Medicine, United States (Krummel et al., 2000 (link)). Lck-EGFP, a fusion protein of mouse Lck and EGFP via a six aa linker expressed in the pcDNA3 under the CMV-promotor, was from Tony Magee, Imperial College London, United Kingdom (Janes et al., 1999 (link)). Borosilicate glass (1B100F-4) was from World Precision instruments (Sarasota, FL, United States).
7
Lipid Labeling for Cellular Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fatty acids were from TCI America (Portland, OR, USA), PEG was from Laysan Bio (Arab, AL, USA), and MTz-NHS was from Click Chemistry Tools. DiI-C18:2, and DiI-C16, DiI-C12 were from ThermoFisher (Waltham, MA, USA). DiI-C18 (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindotricarbocyanine Perchlorate) and DiR (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindotricarbocyanine Iodide) were from Biotium (Hayward, CA, USA). DiI-NH2 was synthesized by a previously reported method.[10 (link)] All the lipids were stored as 1 mM stocks in ethanol. Anticoagulant Citrate Dextrose (ACD) buffer was obtained from the Children’s Hospital Colorado Blood Donation Center and kept sterile before use. Goat IgG fraction to mouse C3 and human C3 (Catalog No. 55 463 and 55 033, respectively) was from MP Biomedicals (Solon, OH, USA). ChromPure human and mouse IgG was from Jackson Immuno Research (West Grove, PA, USA). Secondary antibodies IRDye 80°CW labeled donkey antigoat, goat antihuman, and goat antimouse IgG were from LI-COR Biosciences (Lincoln, NE, USA).
8
Preparation and Characterization of Bacillus subtilis Spores
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterial strains, and culture conditions B. subtilis PS832 is a prototrophic 168 laboratory strain. Strain PS4150 is derived from PS832 carries the cotE and gerE deletion mutations and is a coat defective mutant [21] . Strains PS832 SpoVAEa-SGFP2 and PS4150 SpoVAEa-SGFP2 were derived from PS832 and PS4150, respectively. These two strains contain a SpoVAEa-C-terminal SGFP2 fusion, integrated at the spoVAEa locus, and expressed under the control of the spoVA operon′s promoter [35] . Spores of all strains were prepared in 2×SG sporulation medium at 37 °C as described previously [36] . If required, the fluorescent dyes DiIC 12 (1 μg/ml, ThermoFisher) or FM5-95 (2 μg/mL, ThermoFisher) were added to the sporulation culture, when it reached the peak optical density at 600 nm (OD 600 ). Spores were harvested and purified, including centrifugation through HistodenZ as described previously [16] . Spores (OD600 ~ 60, in MIlliQ water) used in the current work had ≥98% dormant spores, and were essentially free of germinated spores, cells or cell debris as verified by phase contrast microscopy.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!