Jurkat cells (10 × 106) were induced for apoptosis with staurosporine (Sigma-Aldrich, 1 µM/106 cells in 1 ml RPMI medium containing pen/strep/glutamine and 10% FBS) for 5 h. Cells were washed three times in PBS, labeled with CypHer5E (GE Healthcare; 1 µl CypHer5E/1 ml serum-free medium) for 30 min, and washed twice with PBS. Peritoneal macrophages were isolated as described, and 150 × 103 cells were plated on an eight-well glass chamber slide (Nunc) and fed with 750 × 103 pre-labeled apoptotic Jurkat cells for 4 h in a total of 150-µl medium with or without soluble PROS1 (25 nM; from Enzyme Research Laboratories). Next, the medium was aspirated, and bound cells were washed gently with PBS. Adherent cells were subsequently fixed in 200 µl of 4% PFA; 5% sucrose for 15 min. Cells were washed in PBS and incubated with 200 µl phalloidin (5 U/ml, CF488A conjugate, Biotium) at 4°C overnight. Then, cells were washed three times with PBS (10 min each) and stained in 200 µl of Hoechst 3570 (20 µg/ml, Invitrogen) for 5 min, and rinsed with PBS. The chambers were removed, mounted with Fluoromount G, and visualized under a Nikon A1 confocal microscope. The number of CypHer5E+ engulfed ACs per macrophage was scored.
Cypher5e
Manufactured by GE Healthcare
Sourced in United States
CypHer5E is a pH-sensitive fluorescent dye that can be used to measure pH levels in various biological applications. It is designed to emit fluorescence in response to changes in pH, allowing researchers to monitor pH dynamics in real-time.
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Lab products found in correlation
Celltrace violet, by Thermo Fisher Scientific (4 mentions)
Anti mouse igm f ab 2, by Jackson ImmunoResearch (2 mentions)
Biotinylated anti cd23, by BD (2 mentions)
Il 21, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Corning (2 mentions)
Rpmi 1640 medium, by Corning (2 mentions)
Non essential amino acids (neaa), by Corning (2 mentions)
Streptavidin microbeads, by Miltenyi Biotec (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Annexin 5 recombinant protein, by Thermo Fisher Scientific (2 mentions)
Tamra, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Biotin, by Merck Group (1 mentions)
Alexa488 cholera toxin b, by Thermo Fisher Scientific (1 mentions)
Lipidtox red, by Thermo Fisher Scientific (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Protease inhibitor cocktail p1860, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Streptavidin, by Merck Group (1 mentions)
16 protocols using cypher5e
1
Phagocytosis of Apoptotic Cells by Macrophages
2
Fluorescent Labeling of Adipocytes
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Adipocytes were labeled using succinimidyl esters of AlexaFluor (Alexa)546 and Alexa488 (Invitrogen), FITC, biotin (Sigma-Aldrich, St. Louis, MO), or CypHer 5E (GE Healthcare, Chalfont St. Giles, UK). Alexa546-biocytin, Alexa488-cholera toxin B (CtB), and LipidTOX-Red were purchased from Invitrogen. Streptavidin, bafilomycin A1, and protease inhibitor cocktail (P1860), containing aprotinin, bestatin, E-64, leupeptin, and pepstatin A, were purchased from Sigma-Aldrich.
3
Efferocytosis Assay for B Cells
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CD23+ B cells were purified from single cell suspensions of splenocytes with biotinylated anti-CD23 (BD Bioscience; #553137) and streptavidin microbeads (Miltenyi Biotec; #130-048-101) according to manufacturer’s instructions. Cells were cultured for 2–3 d in RPMI 1640 medium (Corning) supplemented with non-essential amino acids (Corning), 2 mM l -Glutamine (Corning), 25 mM HEPES (pH 7.2–7.6), and 50 μM β-Mercaptoethanol and stimulated with 5 μg/mL F(ab’)2 anti-mouse IgM (Jackson ImmunoResearch), 5 μg/mL purified anti-mouse CD40 (BioXcell), and 50 ng/mL IL-21 (Peprotech). For thymocyte engulfment assays68 (link) thymocytes were harvested and isolated from 4–6 week-old WT mice and treated with 50 μM Dexamethasone for 4 h to induce apoptosis. Apoptotic thymocytes were then stained with 1 μM CypHer5E (GE Healthcare) for 45 min at 37 oC in serum-free Hank’s Balanced Sodium Solution (HBSS). Stained thymocytes were co-cultured with splenocytes at a 1:10 splenocyte to thymocyte ratio. Apoptotic thymocytes were removed by washing with cold PBS and splenocytes were assessed for efferocytosis by flow cytometry. MHC-II expression was compared on efferocytic (CypHer5E+) and non-efferocytic (CypHer5E−) B cells.
4
Phagocytosis of Apoptotic Cells Assay
5
Dual Fluorescent Labeling of Bacteria
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Heat-killed bacteria were stained with 5 μM Oregon Green 488-X succinimidyl ester (Invitrogen) at 37 °C with gentle rotation and protected from light for 30 min. This was followed by a change of buffer to sodium carbonate (0.1 M, pH 9.0) and staining with the pH-sensitive dye CypHer5E (General Electric) 20 μg/ml for 2 h in room temperature under gentle rotation in the dark. The samples were washed two times with Na-medium (5 min, 8,000 × g, swing-out rotor). The stock of labeled bacteria was stored up to 5 days in fridge protected from light and was used for all phagocytosis experiment. Prior to opsonization bacteria were sonicated until (VialTweeter; Hielscher) any large aggregates of bacteria were disperse, which was confirmed by microscopy. Staining was checked by flow cytometry (CytoFLEX, Beckman-Coulter), for CypHer5E, 1 μl of sodium acetate (3 M, pH 5.0) was added to confirm pH-sensitivity.
6
Pneumococcal Infection Macrophage Response
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At day 21 post-IAV infection, log-phase S. pneumoniae was labeled with the pH-sensitive dye Cypher5e (GE Healthcare Life Sciences) for 30 min at room temperature. Approximately 107 CFU of labeled bacteria were administered intranasally. The inoculum was confirmed by growth on blood agar plates. Four hours later, BAL was performed, cells were stained with F4/80 (PeCy-7), and flow cytometry was performed. Results are presented as the frequency of Cypher5e+ macrophages (F4/80+) among total cells.
7
Lack Antigen Immunofluorescence Staining
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The Lack antigen was produced and purified by the Recombinant Protein platform (UMR144, Institut Curie, Paris, France). Lack peptide (aa 156-173) was synthetized by PolyPeptide Group. LPS from Salmonella enterica serotype typhimurium and recombinant rat Galectin-8 were purchased from Sigma-Aldrich. Cypher5E was purchased from GE Healthcare.
The following primary antibodies were used for immunofluorescence: rabbit anti-γ-Tubulin (Abcam, #Ab11317, 1/1000), rat anti-Lamp-1 (CD107a; BD PharMingen, #553792, 1/400), polyclonal rabbit anti-OVA (1/500), AlexaFluor647-conjugated rat anti-CD169 (AbD Serotec, #MCA974A647, 1/50). The following secondary antibodies were used: Cy3-conjugated F(ab’)2 donkey anti-rabbit (Jackson ImmunoResearch, 1/200) and AlexaFluor488- and 647-conjugated donkey anti-rat (Life Technologies, 1/200). 4’,6-Diamidino-2-phenyindole (DAPI, Sigma Aldrich, 1/5000) was used to counterstain nuclei.
The following primary antibodies were used for immunofluorescence: rabbit anti-γ-Tubulin (Abcam, #Ab11317, 1/1000), rat anti-Lamp-1 (CD107a; BD PharMingen, #553792, 1/400), polyclonal rabbit anti-OVA (1/500), AlexaFluor647-conjugated rat anti-CD169 (AbD Serotec, #MCA974A647, 1/50). The following secondary antibodies were used: Cy3-conjugated F(ab’)2 donkey anti-rabbit (Jackson ImmunoResearch, 1/200) and AlexaFluor488- and 647-conjugated donkey anti-rat (Life Technologies, 1/200). 4’,6-Diamidino-2-phenyindole (DAPI, Sigma Aldrich, 1/5000) was used to counterstain nuclei.
8
Tau Antibody Characterization and Labeling
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In this study we used tau antibodies, 6B2 and 4E6, that have been generated against the Tau386-408[P-Ser396, 404] region. These IgG1κ mouse antibodies were previously characterized by our laboratory (7 (link), 12 (link), 14 (link), 16 (link), 19 (link), 22 (link), 23 (link), 27 (link)). As a control, a non-specific mouse IgG1κ (IgG1, eBioscience) antibody was used. The antibodies were tagged with Cypher5E (GE Healthcare) fluorescent marker where mentioned. Cypher5E is a pH sensitive dye, and only fluoresces within acidic compartments, like endosomes or lysosomes. The PHF-enriched brain fraction was tagged with Alexa Fluor 488 (Invitrogen) where mentioned. All tagging was performed as outlined in the manufacturer's instructions.
9
Bispecific Antibody Internalization Assay
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The CD34 x G6B bispecific and a non-targeting isotype control (DNP-MB273, Acrobiosystems) were conjugated to CypHer5E (GE Healthcare Life Sciences), a red-excitable, pH-sensitive cyanine dye detected in the APC or Ax647 channel that maximally fluoresces at an acidic pH (i.e., after movement from a receptor on the cell surface to acidic endosomes upon internalization). HEL and SET-2 cells were re-suspended in serum-free, no phenol red RPMI and incubated with DMSO or inhibitors for 30 minutes at 37°C, 5% CO2 prior to the addition of either the CD34 x G6B bispecific or isotype control (5 μg/ml), followed by a further incubation for 30 minutes at 37°C, 5% CO2. For flow cytometry, cells were then washed twice with PBS and re-suspended in PBS for flow analysis on a Beckman Flow CytoFLEX cytometer. For live cell imaging, cells were plated onto slides with flat wells (Hendley-Essex) and imaged on a Zeiss inverted confocal LSM870 with an apochromatic 40X oil immersion objective lens. Two inhibitors were used - Dynasore, a GTPase inhibitor of dynamin (Sigma) (Macia et al., 2006 (link)) at 100 μM and Pitstop 2 (Sigma) (von Kleist et al., 2011 (link)) at 30 μM concentration, that inhibits the clathrin terminal domain as well as clathrin-independent endocytosis.
10
Apoptosis Induction and Phagocytosis Assay
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For induction of apoptosis, human Jurkat T cells resuspended in RPMI with 1% BSA were treated with 150 mJ/cm2 ultraviolet C irradiation (Stratalinker) and incubated for 4h at 37 °C, 5% CO2. For antibody-dependent phagocytosis, Jurkat cells were labeled with αCD3 (25μg/ml, Clone: OKT3 BioLegend) along with Annexin V recombinant protein (to block efferocytosis of any residual dying cells) (3μg/ml, eBioscience) for 1h at 4 °C and the cells were then stained with CypHer5E (GE Healthcare, PA15401) or TAMRA (Invitrogen, C-1171) before use in the engulfment assays. Chines hamster LR73 cells or murine macrophages were seeded in a 24 well plate and incubated with targets at a 1:10 phagocyte to target ratio for the indicated times. Targets were then washed with PBS. Where indicated, phagocytes were rested in culture medium for an additional period of time. Cells were dissociated from the plate with trypsin and the phagocytes were assessed by a flow cytometry-based assay or used in their analysis of RNA or protein23 ,24 . Phalloidin staining was conducted according to the manufacturer’s instruction (Invitrogen). When primary macrophages were used as phagocyte, there is an inherent difference in the absolute % uptake of corpses between experiments performed on different days. Therefore, phagocytic index was used to better compile data from multiple experiments.
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