Sucrose, glucose, HEPES (4–2(2-hydroxymethyl)-1-piparazineethane-sulfonic acid), sodium chloride, and hydrochloric acid (HCl) were purchased from Fisher Scientific. Ammonium sulfate, sodium phosphate, potassium phosphate, potassium chloride, sodium bicarbonate, lectin protein from Canavalia ensiformis, ATTO-488 NHS-ester, TCEP (tris(2-carboxyethyl) phosphine hydrochloride), and doxorubicin were purchased from Sigma-Aldrich. Fetal bovine serum (FBS), trypsin, penicillin, streptomycin, L-glutamine, PBS (phosphate buffered saline), and DMEM (Dulbecco’s modified Eagle medium) were purchased from GE Healthcare. Texas Red-DHPE (Texas Red-1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine) was purchased from Thermofisher. Trypan blue was purchased from Life Technologies. DPPC (1,2 – dipalmitoyl-sn-glycero-3-phosphocholine), DOPC (1,2 dioleoyl-sn-glycero-3-phosphocholine), cholesterol (from ovine wool), DOTAP (1,2 dioleoyl--3-trimethylammonium-propane), and PEG2000-DPPE (1,2 dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000]) were all purchased from Avanti Polar Lipids (Alabaster, AL). LIVE/DEAD fixable violet dead cell stain kit was purchased from Thermo Fisher Scientific. All reagents were used without further purification.
Atto 488 nhs ester
Manufactured by Merck Group
Sourced in United States
The Atto 488 NHS ester is a fluorescent dye used for labeling biomolecules, such as proteins, peptides, and nucleic acids. It has an excitation maximum at 501 nm and an emission maximum at 523 nm. The NHS ester group allows for covalent attachment of the dye to primary amine groups on the target biomolecule.
Automatically generated - may contain errors
Lab products found in correlation
Hepes, by Thermo Fisher Scientific (3 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (3 mentions)
Texas red dhpe, by Thermo Fisher Scientific (2 mentions)
Indo 1 am, by Thermo Fisher Scientific (2 mentions)
Lsr 2 uv, by BD (2 mentions)
Anti hamster ig, by Jackson ImmunoResearch (2 mentions)
Anti cd3ε clone 145 2c11, by Jackson ImmunoResearch (2 mentions)
Cy3 nhs ester, by GE Healthcare (2 mentions)
Alexa fluor 647 donkey anti goat, by Thermo Fisher Scientific (2 mentions)
Alexa fluor donkey anti mouse, by Thermo Fisher Scientific (2 mentions)
Superdex 75 10 300 gl size exclusion column, by GE Healthcare (2 mentions)
Triton x 100, by Thermo Fisher Scientific (2 mentions)
Edta free protease inhibitor tablet, by Merck Group (2 mentions)
Neutravidin, by Thermo Fisher Scientific (2 mentions)
Atto 594 nhs ester, by Merck Group (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Live dead fixable violet dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Thermo Fisher Scientific (1 mentions)
31 protocols using atto 488 nhs ester
1
Lipid Nanoparticle Preparation and Characterization
2
Fluorescent Labeling of Red Blood Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ATTO 488 NHS ester (Sigma-Aldrich), dissolved in PBS, is added to the RBCs to a final concentration of 0.15 mM and incubated at room temperature under continuous stirring. After 30 min, the RBCs were washed three times in the described buffer by centrifugation to remove excess dye.
3
Characterization of Immune Checkpoint Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monoclonal antibodies mouse anti-PD-1, rabbit anti-PD-L1, and mouse anti-CTLA-4 were purchased from Abcam (catalogue numbers ab52587, ab205921, and ab19792, respectively). Polyclonal rabbit anti-CD80 was purchased from MyBioSource (catalogue number MBS2522916). AffiniPure F(ab’)2 fragment donkey anti-mouse IgG (H+L) and peroxidase AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG (H+L) were purchased from Jackson Immuno Research (catalogue numbers 715-006-150 and 711-036-152, respectively). Pierce endogenous peroxidase suppressor, TSA SuperBoost kit, and a Prolong Glass antifade mount were purchased from Thermofisher Scientific (catalogue numbers 35000, B40925 and P36980 respectively). Sodium borohydride, ATTO488 NHS ester, bovine serum albumin, and rhodamine B were purchased from Sigma Aldrich (catalogue numbers 41698-1MG-F, A2153-100G, and 234141-10G, respectively).
4
Nanostructured Protein Production in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nanostructured proteins were produced in E. coli following the method described in Torrealba et al. (9 (link)) and Thwaite et al. (12 (link)). In short, E. coli transformed with the plasmid of interest was cultured in LB with the appropriate antibiotic and recombinant protein expression was induced at OD550nm 0.5–0.8 with 1 mM IPTG (Panreac, Barcelona, Spain). IBs were isolated after 3 h additional incubation at 37°C via enzymatic and mechanical disruption of the cells according to Torrealba et al. (10 (link)), followed by sterility monitoring (12 (link)). Purified nanoparticles, named here IBfrg16G−VHSV, IBTNFα and IBiRFP [an inclusion body made of a non-immunogenic phytochrome-based near infra-red fluorescent protein (iRFP) with the excitation/emission maxima at 690/713 nm (13 (link))], were stored at −80°C until use. Quantification was performed by western blot using an anti-His-tag antibody (Genscript, Piscataway, NJ, USA) and calculating the protein concentration from a standard curve using Quantity One software (Biorad, Hercules, CA, the USA). For flow cytometry or confocal microscopy, IBfrg16G−VHSV and IBTNFα were conjugated with fluorescent Atto-488 NHS ester (Sigma-Aldrich) following manufacturer's instructions.
5
Fluorescent Labeling of Adenovirus-like Particles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The COVID-19 vaccine (Ad26.COV2-S (recombinant)) from Johnson & Johnson was used as a source for adenovirus-like particles encoding the SARS-CoV-2 spike glycoprotein. To fluorescently label the adenovirus-like particles, the buffer was stepwise replaced by borate buffer by using Proteus X-spinner 2.5 Ultrafiltration Concentrator Columns with a molecular weight cutoff of 100 kDa (Serva, catalog no. 42234.01) in five subsequent centrifugation steps (each 20 min, 4 °C, 25,000g). Then, an Atto488 NHS ester (Sigma-Aldrich, catalog no. 41698) was reacted in a 100-fold molar excess with the adenovirus-like particles in borate buffer for 1 h at room temperature. To remove nonbound fluorescent dye, fresh Proteus X-spinner 2.5 Ultrafiltration Concentrator Columns were used in five subsequent centrifugation steps as described previously. Purified adenovirus-like particles-Atto488 were diluted using the original vaccine supernatant recovered from the initial centrifugation steps. The concentration of the adenovirus-like particles in the vaccine as provided by the manufacturer is given as 8.92 log10 infectious units per 0.5 ml. Fluorescently labeled adenovirus-like particles were diluted in the vaccine supernatant to a final maximum working concentration of 3 × 107–3 × 108, assuming 100% recovery in the filtration steps.
6
TCR Activation and Calcium Flux
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse thymocytes were stained with 8 μg/ml of Atto488-NHS Ester (Sigma) or Cy3-NHS Ester (GE Healthcare) for 20 min at 25 °C. Atto488-labeled and Cy3-labeled thymocytes were then incubated with DMSO and CS at various concentrations, respectively. Cells were then stained for surface markers for 10 min at 25 °C. After stained with Indo-1 AM (Life Technologies) at a final concentration of 2 μM at 37 °C for 30 min, cells were washed and resuspended with HBSS + 2% FBS. Anti-CD3ε (clone 145-2C11), a final concentration of 3 μg/ml, and anti-Hamster Ig (Jackson Immunoresearch), a final concentration of 8 μg/ml, were added to cells for crosslinking TCRs. Calcium flux data were acquired with LSR II UV (BD Biosciences). The data were analyzed with FlowJo software (Treestar) by comparing the ratio of emission of Indo-1 on the Indo-1 Blue (450/50 nm) channel to the Indo-I Green (525/50 nm) channel.
7
Unroofed Cell Immunolabeling with Antibodies
8
Reconstitution of Membrane-Bound Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
POPC and DGS-NTA-Ni were purchased from Avanti Polar Lipids Inc. Texas Red–DHPE, NeutrAvidin, tris-HCl (tris hydrochloride), Hepes, IPTG (isopropyl-β-d -thiogalactopyranoside), b-mercaptoethanol, TCEP [tris(2-carboxyethyl)phosphine], and Triton X-100 were purchased from Thermo Fisher Scientific. Tryptone, yeast extract, NaCl, NaH2PO4, Na2HPO4, urea, sodium tetraborate, EDTA, CaCl2, glycerol, EDTA-free protease inhibitor tablets, imidazole, PMSF (phenylmethanesulfonylfluoride), PLL (poly-l -lysine), Atto 594 NHS-ester, and Atto 488 NHS-ester were purchased from Sigma-Aldrich. DP-EG10-biotin was provided by D. Sasaki from Sandia National Laboratories, Livermore, CA (40 (link)). Amine-reactive polyethylene glycol [mPEG-succinimidyl valerate (SVA); molecular weight (MW), 5000] and PEG-biotin (Biotin-PEG SVA; MW, 5000) were purchased from Laysan Bio Inc. All reagents were used without further purification.
9
Fluorescent Labeling of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Highly purified FGFR2 and PLCγ1 proteins (in 20 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mM TCEP) were prepared in 100 mM NaHCO3 buffer (pH 8.3) at 2 mg/ml and labelled with Atto-488 NHS ester or Atto-647 NHS ester (Sigma) respectively and incubated at room temperature for 1 hr (fluorophore to protein molar ratio was 1:1). Highly purified SHP2 proteins (in 20 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mM TCEP) were directly labelled with NTA Atto-550 at room temperature for 10 minutes (fluorophore to protein molar ratio was 1:1). For untagged SHP2 labelling (used in support lipid bilayers experiment) highly purified SHP2 proteins (in 20 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mM TCEP) were prepared in 100 mM NaHCO3 buffer (pH 8.3) at 0.5 mg/ml and labelled with Atto-594 NHS ester and incubated at room temperature for 20 minutes to reduce non-specific labelling that affect the SH2 domain binding ability. Excess dye was removed using G-15 desalting chromatography. Proteins were concentrated and labelling efficiency was measured by Nanodrop 2000 (ThermoFisher).
10
Unroofed Cell Immunolabeling with Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fixed unroofed cells were rinsed with PBS and placed into blocking buffer (PBS with 3 % w/v bovine serum albumin, BSA) for one hour. The unroofed cells were then incubated for one hour in blocking buffer containing 2 μg/mL primary antibody (R-20, Santa Cruz Biotechnology or X22, ThermoScientific). The sample was rinsed with blocking buffer prior to secondary antibody labeling for 30 min (2 μg/mL Alexa Fluor 647 donkey anti-goat, Invitrogen A31571; Alexa Fluor donkey anti-mouse, Invitrogen A21447; or Atto 488 donkey anti-mouse) in blocking buffer. Atto 488 donkey anti-mouse was created with Atto 488 NHS ester (Sigma, 4-molar excess) and unlabeled donkey anti-mouse IgG (Abcam ab6707) and purified through a Superdex 75 10/300 GL size exclusion column (GE Healthcare). Finally, the sample was washed with blocking buffer, PBS, and post-fixed for 20 min. For immunolabeling intact cells, 0.2% Triton X was added to the blocking buffer, and cells were incubated for two minutes in permeabilization buffer (PBS 3 % w/v BSA, 0.5% Triton X) prior to blocking.
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!