Splenocytes were cultured for at least 2 hours and stained for viability with Aqua Amine Live/Dead (Invitrogen) for 30 mins. Following washing and resting for 30 minutes, splenocytes were stimulated with IFNα (PBL Interferon Source, 1000 U/ml), IFNγ (Peprotech, 50 ng/ml), IL-4 (Peprotech, 50 ng/ml), IL-12 (Peprotech, 50 ng/ml), IL-21 (Peprotech, 50 ng/ml), or IL-27 (R&E Systems, 50 ng/ml) for 20 minutes at 37°C. Stimulation was halted with 1.6% paraformaldehyde for 10 minutes at room temperature. Following washing with FACS buffer (0.5% Bovine Serum Albumin and 0.09% Sodium Azide in PBS), splenocytes were permeabilized using pre-chilled MeOH for 20 minutes at 4°C. Following washing, cells were stained for 1 hour at room temperature with fluorophore-conjugated surface antibodies: CD3 (17A2) eFuor450, CD4 (RM4–5) Alexa 700, CD8a (53–6.7) PerCP-Cy5.5, CD19 (1D3) PE-Cy7, NKp46 (29A1.4) APC (all surface antibodies were purchased from eBioscience except NKp46, which was purchased from BioLegend) in addition to intracellular antibodies against phospho-proteins Stat1 Y701 (4A) Alexa 488, Stat3 Y705 (4) Alexa 647, Stat4 pY694 (38) PE, Stat5 Y694 (47) Alexa 488, and Stat6 Y641 (J71–773.58.11) Alexa 647 (BD Biosciences). Following washing, cells were fixed with 1.6% paraformaldehyde and analyzed using an LSR II (Becton Dickinson).
Alexa 647
Manufactured by BD
Sourced in United States
Alexa Fluor 647 is a fluorescent dye used for labeling and detection in various life science applications. It has an excitation maximum at 650 nm and an emission maximum at 665 nm, making it suitable for detection in the red region of the visible spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488, by BD (2 mentions)
Fluorescein isothiocyanate (fitc), by BD (2 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Facsjazz, by BD (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Cell strainer, by BD (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Percp cy5, by Thermo Fisher Scientific (1 mentions)
Il 21, by Thermo Fisher Scientific (1 mentions)
Il 12, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Flowjov, by Tree Star (1 mentions)
Apc cy7, by Thermo Fisher Scientific (1 mentions)
Apc cy7, by BD (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Ps peg cooh, by Polymer Source (1 mentions)
Pmypt, by Cell Signaling Technology (1 mentions)
Volocity software 6, by PerkinElmer (1 mentions)
Fluoromount, by Merck Group (1 mentions)
7 protocols using alexa 647
1
Phosphorylation of STAT Proteins in Splenocytes
2
Quantifying Pluripotency Markers in Cells
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Cells grown on coverslips in Petri dishes or transferred into suspension (106 cells) were fixed with 4% formaldehyde (10 min at 4 °C), washed 2 times with PBS, permeabilized with 0.5% Triton-X100 (10 min at 20 °C), blocked with 3% BSA (15 min at 20 °C), incubated with primary monoclonal mouse antibodies to the OCT4A isoform labeled with Alexa647 (BD Pharmingen, San Diego, CA, USA), diluted with 1% BSA 1:20 (1 h at 20 °C) and washed 3 times with PBS. Negative control samples were supplemented with 1% BSA instead of antibodies. Samples for confocal microscopy were mounted in a solution of DAPI and VECTA SHIELD, and samples for flow cytometry were diluted in 300 μL PBS. Analysis was performed on a Leica TCS SP5X confocal scanning microscope at 60× magnification (Mannheim, Germany) and a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). All experiments were performed at least three times.
3
Detecting SOX10-NL Expressing Cells
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Cultured cells were dissociated with TrypLE select (GIBCO) at 37°C for 5 min for detecting SOX10-NL expression. They were incubated sequentially with antibody against human CD271 (NGFR, p75NTR) conjugates with Alexa647 (BD; diluted 1/100) for 30 min on ice [21 (link)]. Dissociated cells were resuspended with 1% bovine serum albumin in PBS. Cell debris was eliminated with a cell strainer (BD; 35 μm) and suspensions were stained with SYTOX Red stain (Molecular Probes) to exclude dead cells. Cells were analyzed and collected by cell sorter using FACSJazz (BD).
4
Flow Cytometric Analysis of Microglial P2Y12 Expression
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Human microglia were collected by gently scraping and blocked in FACS buffer supplemented with 10% normal human serum and normal mouse IgG (3 μg/mL). Cells were then incubated at 4°C for 30 minutes with either a control isotype antibody directly conjugated with Alexa647 (BD Biosciences, Mississauga, Ontario, Canada) or a polyclonal rabbit anti-P2Y12 antibody (1:100) followed by an anti-IgG1-Alexa647 (1:100). After washing, cells were fixed in 1% formaldehyde and flow cytometry was performed using a FACSCalibur (BD Biosciences).
5
Characterization of CTL Dysfunction Markers
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To characterize potential markers associated with CTL dysfunction in response to LRA-reactivated cells, we exposed CTL1 and CTL2 to cognate HIV-1 antigen over nine weeks. To do so, we stimulated CTL with a 1:1 mixture of irradiated autologous B-cell lines (BCLs) pulsed with cognate HIV peptide (10 μg/ml) and irradiated PBMCs from three healthy HIV-seronegative donors weekly. We used flow cytometry to evaluate variations in the expression of the inhibitory receptors PD-1, TIM-3, LAG-3, and the immune-metabolic marker CD39. Briefly, cells were taken from the culture at weeks 0, 5, 7, and 9 and stained with a Live/Dead probe (APC-Cy7, Invitrogen) and surface markers CD3 (Alexa700, BD Biosciences), CD4 (APC-Cy7, BD Biosciences), CD8 (V500, BD Biosciences), PD-1 (BV421, BD Biosciences), TIM-3 (Alexa 647, BD Biosciences), LAG-3 (PE, BD Biosciences), and CD39 (FITC, BD Biosciences) and incubated at room temperature for 25 min. Samples were washed twice with 1X PBS, fixed in 1% formaldehyde, and acquired on an LSR Fortessa. Data were analyzed with FlowJoV (Tree Star Inc.). Patterns of co-expression of PD-1, LAG-3, TIM-3, and CD39 were analyzed using Pestle and SPICE v5 software (55 (link)).
6
Photophysical Characterization of Polymers
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All of the chemicals and solvents were purchased from Sigma-Aldrich (St Louis, MO, USA) unless indicated otherwise. The fluorescent semiconducting polymer CN-PPV (molecular weight (MW) 15,000, polydispersity 5.9) was purchased from ADS Dyes, Inc. (Quebec, Canada). The photochromic quencher BTE was purchased from TCI (Portland, OR, USA). A comb-like polymer—polystyrene grafted with ethylene oxide functionalized with carboxyl groups (PS-PEG-COOH, main chain MW 8,500, graft chain MW 1,200, total chain MW 21,700, polydispersity 1.25)—was purchased from Polymer Source Inc. (Quebec, Canada). The fluorochromes, including FITC, APC, Alexa 488 and Alexa 647, used in the photobleaching experiment were purchased from BD Bioscience (San Jose, CA, USA). HEPES, EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and streptavidin were purchased from Invitrogen (Eugene, OR, USA). All chemicals were used as received without further purification. High purity of MilliQ water (18.2 MΩ cm−1) was used throughout the experiment.
7
Immunofluorescence Staining of Knee Cells
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Cells were harvested from knee wash and cytocentrifuged (Cytospin; Shandon Lipshaw Inc., Pittsburgh, PA, USA) in cells coverslips. Next, cells were fixed in 4% paraformaldehyde for 20 min at room temperature. Cells were permeabilized for 10 min with triton-X-100 1% and then incubated with primary antibodies p-MYPT (Cell Signaling Technology, Danvers, MA, USA) overnight. The secondary antibody used was anti-rabbit Alexa-647 BD, USA) and nuclei were stained with DAPI (1:1000–BD). Finally, coverslips were prepared with fluoromount (Sigma Aldrich, USA) for confocal microscopy analysis. Images were obtained using the Ti microscope with laser confocal C2 equipped with three different lasers (excitation 405, 488 and 543 nm) and emission filters 450/50 nm (channel 1), 515/30 nm (channel 2) and 584/50 nm (channel 3). At least 500 cells per glass slide were counted and the fluorescence intensity was measured off-line using Volocity software 6.3 (Perkin-Elmer, Waltham, MA, USA).
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