Monoclonal antibodies used for flow cytometry were from: Biolegend; anti-CD11c (N418), Vβ11 (KT11), anti-IAb (AF6-120.1); from ThermoFisher: anti-CD4 (GK1.5 and RM4-5), anti-CD86 (GL1), anti-CD40 (1C10), anti-Foxp3 (FJK-16s), anti–IL-17 (ebio17B7), anti-IFN-γ (XMG1.2), anti-CD11b (M1/70), anti-CD11c (N418), anti-TCRβ (H57-597), anti-ICOS (C398.4A), anti-CD103 (2E7), anti CD16/32 (93), anti-CD25 (PC61.5), anti-ICOSL (HK5.3), anti-GM-CSF (MP1-22E9), Fixable Viability dye eFluor780, CellTrace Violet cell proliferation; from BD: anti-CD25 (PC61), anti-CD3 (145-2C11), and anti–IFN-γ (XMG1.2), anti-Sema4a (5E3/SEMA4A). For flow cytometry analysis, single cell suspensions were incubated with FcBlock (anti-CD16/32 FcγRII-RIII) for 10 min, at 4°C and stained with antibodies. Intracellular cytokine stainings were done using the Intracellular Fixation & Permeabilisation buffer set (eBioscience). For IFN-γ, IL-17, and GM-CSF staining, cells were first re-stimulated in complete RPMI containing PMA/ionomycin, and incubated 4 h at 37°C, 5% CO2. Golgi stop solution (BD Biosciences) was added to the last 2.5 h of culture. Data were acquired with an Attune NxT (Life Sciences) or a Gallios (Beckman Coulter) and analyzed using FlowJo software (FlowJo company).
Golgistop solution
Manufactured by BD
Sourced in United States
GolgiStop solution is a chemical agent used in biological research to disrupt the Golgi apparatus within cells. It functions by preventing the transport of proteins from the endoplasmic reticulum to the Golgi complex, effectively halting the normal secretory pathway. The solution is designed for use in cell culture and in vitro experiments to investigate cellular processes related to the Golgi apparatus.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm plus kit, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Perm wash buffer, by BD (3 mentions)
Anti isotype controls, by Exbio (3 mentions)
Facscalibur, by BD (2 mentions)
Fix and perm, by Thermo Fisher Scientific (2 mentions)
Facsaria 2, by BD (2 mentions)
Elisa kit, by R&D Systems (2 mentions)
Cytofix cytoperm solution, by BD (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (2 mentions)
Cytofix buffer, by BD (2 mentions)
Anti kir2dl2 2ds2 2dl3 cd158b pe, by Thermo Fisher Scientific (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Anti ifn γ xmg1.2, by BD (1 mentions)
Intracellular fixation permeabilisation buffer set, by Thermo Fisher Scientific (1 mentions)
Anti cd107a antibody clone h4a3, by BioLegend (1 mentions)
Fix perm buffer, by BD (1 mentions)
27 protocols using golgistop solution
1
Multiparameter Flow Cytometric Analysis
2
NK Cell-Mediated Neuroblastoma Killing
3
Kidney Cell Cytokine Profiling
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We prepared and stained single-cell suspensions from both kidneys as described previously [11 (link), 15 (link), 39 (link)]. The antibodies used for FACS were as follow: fluorescein isothiocyanate-conjugated rat anti-mouse CD11b (#557396, Becton Dickinson (BD), San Jose, CA, USA), Alexa Fluor 647-conjugated rat anti-mouse F4/80 (#565853, BD), phycoerythrin-conjugated rat anti-mouse TNF-α (#554419, BD), and phycoerythrin-conjugated rat anti-mouse IL-10 (#554467, BD). To examine intracellular cytokine expression, kidneys were incubated in RPMI 1640 containing GolgiStop™ solution (#554715, BD) for 8 h in 5% CO2 at 37°C. The cells were washed in phosphate-buffered saline, suspended in FACS buffer (#554656, BD) with protein-block (Fcg III/II Receptor, 1:100 dilution, BD, #553141), and incubated with cell surface markers for 30 min on ice. Then, the cells were fixed and permeabilized using Fix/Perm buffer (#554715, BD). After the staining of intracellular cytokines, the cells were washed twice in Fix/Perm buffer, and suspended in FACS buffer to allow the resealing of the permeabilized membranes. We collected 1.0 × 105 to 5.0 × 105 total kidney cells using FACSCalibur (BD), and analyzed the data using FlowJo software 9.3 (Tree Star, Palo Alto, CA, USA).
4
Characterizing T-cell Responses in Murine Arthritis
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The inguinal lymph nodes (LNs) from each treated group were prepared. Briefly, on experimental day 42, inguinal LN cells were extracted from each mouse using mechanical disruption with mesh, and single-cell suspensions were obtained. For intracellular detection of cytokines, LN cells (2 × 106) were stimulated with CII (20 ug/ml) for 72 h; Golgi stop solution (BD Biosciences) was added for 4 h before the culture cells were harvested. The cells were then washed twice in FACScan buffer and stained with a phycoerythrin (PE)-conjugated anti-mouse CD4 (Biolegend) Ab. Cells were then fixed and processed for intracellular staining using the Cytofix/Cytoperm Plus Kit (BD Biosciences) according to the manufacturer’s instructions. The FITC-conjugated mAbs specific to murine IFN-γ, IL-17A, and Foxp3 were purchased from BioLegend. All samples were detected using an Accuri 5 flow cytometer, and the mean fluorescence intensity was calculated using a C6 Accuri system software (Accuri Cytometer)
5
Flow Cytometric Analysis of HLA Tetramers and Intracellular Cytokines
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All flow cytometric measurements were performed on a FACSCanto II (BD) and evaluated using FlowJo version 9.2 (Tree Star, Ashland, OR, USA). For HLA tetramer analysis, T cells were washed in FACS buffer containing 2% FCS, 2 mM EDTA (Carl Roth, 8040) and 0.02% sodium azide (Merck Millipore, 8223350100) in PBS, followed by a staining for dead cells using Live/Dead fixable Aqua dead cell stain kit (Invitrogen, L34957). Afterwards, cells were washed and incubated with 2.5 µg/mL of the respective tetramer in tetramer solution (FACS buffer with 50% FCS) for 30 min at RT. Finally, cell surface molecules were stained with CD8-PerCP (Biolegend, 301030) for 20 min at 4°C.
Intracellular cytokine staining was performed by incubating in vitro primed T cells with 10 µg/mL candidate or control peptide, Golgi-Stop-Solution (BD Bioscience, 554724) and anti-CD107a-FITC antibody (BD Bioscience, 555800) for 6 h. Afterwards, cells were stained for dead cells using Live/Dead fixable Aqua dead cell stain kit followed by staining with CD8-PerCP for 20 min. Intracellular staining was performed after 30 min permeabilization with Cytoperm/Cytofix solution (BD Bioscience, 554722) using the following antibodies: IFNγ-PE-Cy7, IL-2-APC, TNFα-PacificBlue and MIP-1β-PE (all BD Bioscience, 557844, 554567 and 550078; except TNFα, Biolegend, 502920).
Intracellular cytokine staining was performed by incubating in vitro primed T cells with 10 µg/mL candidate or control peptide, Golgi-Stop-Solution (BD Bioscience, 554724) and anti-CD107a-FITC antibody (BD Bioscience, 555800) for 6 h. Afterwards, cells were stained for dead cells using Live/Dead fixable Aqua dead cell stain kit followed by staining with CD8-PerCP for 20 min. Intracellular staining was performed after 30 min permeabilization with Cytoperm/Cytofix solution (BD Bioscience, 554722) using the following antibodies: IFNγ-PE-Cy7, IL-2-APC, TNFα-PacificBlue and MIP-1β-PE (all BD Bioscience, 557844, 554567 and 550078; except TNFα, Biolegend, 502920).
6
Quantifying Intracellular Protein Levels
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MCF7 cells were incubated (30 minutes at 4°C) with each antibody following treatment for 12 hours with GolgiStop solution (BD Biosciences, Mississauga, ON, Canada) and cell permeabilization with Inside Stain Kit (Miltenyi, Bergisch Gladbach, Germany). Cells were analyzed with a FACS Calibur cytofluorimeter using CellQuest software (BD Biosciences). 104 events for each sample were acquired in all analyses [44 (link)].
7
NK Cell Cytotoxicity Assay
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In vitro cytotoxicity experiments were performed using K562 or Beas-2B cells as the target and NK cells as effector cells. NK cells were added to K562 or Beas-2B cells with a 5:1 effector:target ratio [23 (link)]. NK cell degranulation was evaluated by CD107a staining (anti-CD107a-PE; clone H4A3; BD Biosciences) after 3 h of treatment with Golgi Stop solution (BD). Labeled cells were analyzed with a FACSCantoII flow cytometer (BD, Milano, Italy) and FlowJo software (Tree Star Inc., Ashland, OR, USA).
8
Activation and Cytokine Profiling of Human Lymphocytes
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Human lymphocytes (1 × 106) were plated in 96-well plates and were cultured in RPMI 1640 complete medium containing 10% FCS (Invitrogen), 2 mM l -glutamine (Invitrogen), 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and recombinant human 10 ng/ml IL-2 (PeproTech, Rocky Hill, NJ). For T and NK cell stimulation, IL-12/IL-18 (50 ng/ml; PeproTech) was supplemented into the medium for 6 h, followed by intermediate FACS analysis to detect cell activation. For measuring the cytokine secretion, the cultured cells were collected and analyzed by FACS after addition of Golgi stop solution (BD Bioscience) for at least 6 h.
9
Th1 and Th2 Cytokine Profiling
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Naïve CD4 T cells were activated, transduced as above, and differentiated into Th1- or Th2-polarizing conditions for 3 days. The Th1 and Th2 cells were then re-stimulated for 4 h with 1 μM ionomycin (Sigma-Aldrich) and 10 nM phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich) with Golgi stop solution (BD, Franklin Lakes, NJ, USA). For intracellular cytokine staining, Th1/Th2 cells were fixed/permeabilized by using a BD intracellular cytokine staining kit for 20 min. The fixed cells were stained with anti-IL-4-antibody-phycoerythrin conjugate (anti-IL-4-PE) (504104, BioLegend, San Diego, CA, USA), anti-IL-5-PE conjugate (eBioscience, 12-7052-82), anti-IL-13-PE conjugate (eBioscience, 12-7133-82), or anti-interferon (IFN)-γ-PE (BioLegend, 505807) for 30 min at 4°C. The stained Th1/Th2 cells were washed with phosphate buffered saline (PBS), and cellular fluorescence was determined by using fluorescence-activated cell sorter (BD FACSVantage SE). FACS data were acquired and analyzed by CellQuest™ software (BD Bioscience).
10
Evaluating NK Cell Function by Flow Cytometry
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To examine the function of NK cells, isolated PBMC were co-cultured with or without K562, an NK cell-sensitive cell line, at an effector-to-target (E/T) ratio of 1:1. FITC conjugated anti-human CD107a monoclonal antibody (H4A3; BioLegend) was added at the time of co-culture to assess degranulation. After co-culturing for 1 hour, Golgi stop solution (1 μl/ml; BD Bioscience) was added and the cultures were incubated for 4 hours. Then, a fixation/permeabilization solution (554714, 100 μl/sample, BD Bioscience) was added. After fixation, the samples were stained with PE conjugated anti-human IFN-γ monoclonal antibody (25723.11; BD Bioscience), CD56, CD3, NKp46 and NKG2A antibody and analyzed using flow cytometry.
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