Twenty-four hours before collecting the neural cultures by scraping, the medium was changed to astrocyte-conditioned medium (ScienCell Research Laboratories). SYNs were then isolated as described previously using a synaptic protein extraction reagent (Syn-PER; Thermo Fisher Scientific)20 (link). The resulting SYNs were collected and freeze-thawed (identically prepared and handled), and then pre-assay-labeled with an amine-reactive and pH-sensitive dye (pHrodo Red, succinimidyl ester; Thermo Fisher Scientific, catalog no. P36600) for real-time (live) assays according to the manufacturer’s instructions. For fixed experiments, to show colocalization with synaptic markers (Supplementary Fig. 9 ), SYNs were labeled with the fixable pH-sensitive dye CypHer5E NHS Ester (GE Healthcare Life Sciences, catalog no. PA15401) as described previously33 (link).
Cypher5e nhs ester
Manufactured by GE Healthcare
CypHer5E NHS Ester is a fluorescent dye used for labeling proteins and other biomolecules. It is a succinimidyl ester that can react with primary amines to form stable amide bonds, enabling covalent attachment of the dye to the target molecule.
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Lab products found in correlation
4 protocols using cypher5e nhs ester
1
Synaptic Protein Isolation and Labeling
2
Macrophage Efferocytosis Quantification
3
Measuring Extracellular Albumin Catabolism
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To measure catabolism of extracellular albumin, cells were directly seeded in complete or leucine-free medium supplemented with 3% BSA and cultured overnight (16 h). DQ-BSA Green (0.05 mg/mL) and LysoTracker Red (10 nM; Life Technologies) were added to the cells for 4 h, after which cells were washed once with PBS, dissociated with trypsin, resuspended in serum-containing medium, and pelleted at 400g for 5 min at 4°C. Pelleted cells were resuspended in FACS buffer (PBS, 2% FBS) and samples were analyzed immediately.
Determination of necrotic cell engulfment was achieved by first pelleting 5 × 107 necrotic FL5.12 cells at 700g for 5 min at room temperature and resuspending them in 10 mL of PBS with 1 µM cypHer5E NHS ester (GE Healthcare) for 30 min at 37°C. Stained cells were pelleted again and resuspended in assay medium at a concentration of 2 × 106/mL along with inhibitors or vehicle controls and added to the wells of a tissue culture plate containing target cells. Plates were spun in a swinging bucket rotor centrifuge twice at 500g for 1 min at room temperature, rotating the plate in between spins, and returned to the tissue culture incubator for 2–6 h. Cells were harvested in the same manner as for the DQ-BSA assay above. Samples were analyzed on a BD Fortessa or LSRII flow cytometer. Analysis of data and generation of histograms was performed with FlowJo (Treestar).
Determination of necrotic cell engulfment was achieved by first pelleting 5 × 107 necrotic FL5.12 cells at 700g for 5 min at room temperature and resuspending them in 10 mL of PBS with 1 µM cypHer5E NHS ester (GE Healthcare) for 30 min at 37°C. Stained cells were pelleted again and resuspended in assay medium at a concentration of 2 × 106/mL along with inhibitors or vehicle controls and added to the wells of a tissue culture plate containing target cells. Plates were spun in a swinging bucket rotor centrifuge twice at 500g for 1 min at room temperature, rotating the plate in between spins, and returned to the tissue culture incubator for 2–6 h. Cells were harvested in the same manner as for the DQ-BSA assay above. Samples were analyzed on a BD Fortessa or LSRII flow cytometer. Analysis of data and generation of histograms was performed with FlowJo (Treestar).
4
Oligomeric Aβ Labeling with CypHer5E
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Aβ (1-42) (641-15, California peptide) was monomerized using a previously published protocol (109 (link)), using hexafluoroisopropanol (52517, Sigma-Aldrich). Then, 5 mM monomeric Aβ samples were incubated for 24 h at 4 °C in F12 media to make a 200 μM stock of oligomeric Aβ. Samples were then incubated with CypHer5E-NHS ester (PA15401, GE Healthcare) diluted in 0.1 M sodium bicarbonate for 30 min covered and at room temperature. Following incubation, Biospin columns (7326227, Bio-Rad) were used to quench unbound dye. CypHer5E-tagged Aβ oligomers were stored at 4 °C prior to cell culture treatment or injection (107 (link)).
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