Cells were fixed in 4% paraformaldehyde-PBS solution. For visualization of intracellular markers, cells were permeabilized with 0.1% Triton X-100-PBS solution, blocked with 2% BSA-PBS solution for 1 hr at room temperature, and incubated overnight at 4°C with primary antibodies as listed in Table S4 . Appropriate fluorescence-tagged secondary antibodies (Molecular Probes and Vector Laboratories) were used for visualization. Cells were mounted in DAPI mounting medium (Vector Laboratories), and images were obtained using an Olympus IX71 microscope equipped with a DP71 digital camera or Olympus FSX100 system. MetaMorph Microscopy Automation and Image Analysis Software (Molecular Devices) were used to count positive cells in seven to ten randomly selected images at a final magnification of ×200 from each of three independent experiments. For flow cytometry, cells were dissociated into single cells using GIBCO Cell Dissociation Buffer (Invitrogen) and incubated in 1% BSA-PBS solution with the appropriate antibodies as listed in Table S4 . For unconjugated primary antibodies, the cells were incubated with Alexa-Flour 488-conjugated anti-mouse IgG/IgM (Molecular Probes) for raising suitable secondary antibodies. Flow cytometry was performed using FACSCalibur (BD Biosciences) and analyzed using WinMDI 2.8 or FACSVerse (BD Biosciences) and FlowJo software.
Gibco cell dissociation buffer
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Gibco Cell Dissociation Buffer is a laboratory reagent designed to facilitate the detachment of adherent cells from cell culture surfaces. It is a ready-to-use, calcium and magnesium-free solution that helps break down the cell-to-cell and cell-to-matrix adhesions without affecting cell viability.
Automatically generated - may contain errors
Lab products found in correlation
Flow express software, by De Novo Software (3 mentions)
Silac mem medium, by Thermo Fisher Scientific (2 mentions)
Light l proline, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
L arginine, by Cambridge Isotopes (2 mentions)
Fsx100 system, by Olympus (1 mentions)
Facscalibur, by BD (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Fluorescence tagged secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Dapi mounting medium, by Vector Laboratories (1 mentions)
Metamorph microscopy automation and image analysis software, by Molecular Devices (1 mentions)
Guava easycyte flow cytometry, by Merck Group (1 mentions)
Accuri c6, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Clone is5 21f5, by Miltenyi Biotec (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Fcs express 3, by De Novo Software (1 mentions)
9 protocols using gibco cell dissociation buffer
1
Immunofluorescence and Flow Cytometry Analysis
2
Cell Surface Protein Analysis by Flow Cytometry
3
Cell Surface Marker Quantification
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Cells were detached with enzyme-free Gibco® Cell Dissociation Buffer (Invitrogen) and suspended at a concentration of 5 × 105 cells in 100 μL cold PBS/1% bovine serum albumin (BSA). Primary antibodies or corresponding isotype control antibodies were incubated with cells for 30 min on ice. Following two washes with 1 mL of PBS/1% BSA, fluorescence measurements were collected using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using the Flow Express software (De Novo Software, Los Angeles, CA, USA).
4
Quantifying Cell Adhesion Molecules
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E-selectin and ICAM-1 are molecules associated with cell-to-cell adhesion and are found on cell surfaces. The expression of these proteins was quantified using BD Accuri TM C6 Flow Cytometer (B.D. Biosciences). Cells were detached with enzyme-free Gibco cell dissociation buffer (Invitrogen) and suspended in cold PBS 200 ul of 1% bovine serum albumin (BSA) at a 1×106 cells concentration. The cells were treated with antibodies against each cell adhesion protein, P.E. Mouse Anti-Human CD62P targeting E-selectin and P.E. Mouse Anti-Human CD54 targeting ICAM-1, Anti-Human/Mouse beta-Catenin Alexa Fluor® 488, and Mouse Anti-Human CD144 were incubated with cells for 30 min on ice. Cells were then washed twice with 1 mL PBS/1% BSA, and fluorescence measurements were detected on an Accuri C6 flow cytometer. Analysis of the data was performed using the Flow Express software (De Novo Software, Los Angeles, CA, USA). The flow cytometry assay provides protein assay using live cells under different conditions.
5
SILAC Metabolic Labeling Protocol
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Cells were grown in SILAC MEM medium (Thermo Fisher Scientific) lacking lysine and arginine, supplemented with dialysed 10% FBS (Sigma-Aldrich), 200 mg/l light L-Proline (Sigma-Aldrich) and either heavy L-Lysine (13C615N2; 146 mg/l) and L-Arginine (13C615N4; 84 mg/l) (Cambridge Isotope Laboratories) or their light equivalents (Sigma-Aldrich). Cells were passaged with non-enzymatic Gibco Cell Dissociation Buffer (Thermo Fisher Scientific). >95% heavy amino acid incorporation rate was validated by mass-spectrometry.
6
Flow Cytometry Analysis of Melanoma
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Melanoma cells were plated into 6-well plates and incubated overnight. After washing with DPBS, cells were dissociated using Gibco Cell Dissociation Buffer (Thermo Fisher Scientific, Schwerte, Germany), resuspended in FACS buffer (DBPS, 1% fetal calf serum, 1 mM EDTA), and incubated with the FcR blocking reagent (Miltenyi Biotec, Bergisch-Gladbach, Germany) at 4 °C for 10 min. Thereafter, cells were stained using anti-CD111-APC (Nectin-1, clone R1.302; Miltenyi Biotech; dilution 1:11) or isotype (clone IS5-21F5; Miltenyi Biotech; dilution 1:50), and anti-CD270-PE (HVEM, clone 122; Biolegend; dilution 1:33) or isotype (clone MOPC21; Acris/OriGene, Herford, Germany; dilution 1:33) at 4 °C for 20 min. After fixation with 4% paraformaldehyde, cells were analyzed using FACS Canto II with FACSDiva software for automatic compensation and measurement of samples (BD Biosciences) and FCS Express 3 software (De Novo Software, Los Angeles, CA, USA). Each cell line was analyzed in at least three independent experiments.
7
SILAC Metabolic Labeling Protocol
8
Flow cytometry analysis of ACKR4 mutants
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Transfected HeLa cells were seeded at 2.5 × 105 cells per well in 6 well plates. About 24–36 h post-transfection, cells were washed with FACS buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM sodium phosphate, 5 mM HEPES, pH 7.5) and detached using Gibco Cell Dissociation Buffer (ThermoFisher). Cells were incubated with α-hACKR4 primary antibody (clone 13E11; #362102 Biolegend, dilution 1:750) at 8°C for 40 min followed by intense washing and incubation with goat α-mouse IgG coupled to Alexa647 (#A-21235 ThermoFisher, dilution 1:1000) for additional 20 min. To determine chemokine binding capacities to different ACKR4-EYFP mutants, transfected cells were incubated with 25 nM site specific labeled human CCL19 (CCL19Dy649P1) at 8°C for 30 min. After washing, cells were analyzed by flow cytometry on a LSR II (BD Biosciences). Flow cytometry data were analyzed using FlowJo V10 (BD Biosciences). Medians of chemokine or antibody fluorescence of EYFP+ cells were related to the median of EYFP to consider transfection efficiency.
9
Prostate Cell Culture Dissociation Optimization
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DU145 or 22RV1 prostate cells were cultured in 96-well V-bottom plates (no. 3894; Corning, Corning, NY), at 50,000 cells per well, in 200 µl of cell culture media. Plates were cultured at 37 °C, 5% CO 2 , for either 48 or 72 h. All wells were then stained with a near-infrared Live/Dead stain (Thermo Fisher). Cells were washed in phosphate-buffered saline, then dissociated from the plate using 25 µl per well of StemPro Accutase (Thermo Fisher), TrypLE (Thermo Fisher), 0.5 M EDTA (Invitrogen, Waltham, MA), or Gibco Cell Dissociation Buffer (Thermo Fisher). Cells were then left in Accutase for 5, 10, 15, or 20 min, and shaken for the final 0, 2, or 5 min at 1400 rpm on the iQue instrument. The cell dissociation reagent was then quenched with 25 µl of Qsol (Intellicyt) per well. Plates were shaken at 2200 rpm for 10-15 s prior to the run to resuspend cells. Lastly, samples were acquired on the iQue Screener PLUS (Intellicyt) with the Violet-Red-Blue laser configuration using ForeCyt (version 5.4.6180) with a 10 s sip time (approximately 18 µL). Counts of the live target cells in each well were obtained by negatively gating out dead cells and debris with the nearinfrared Live/Dead stain.
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