The Jurkat T cells were obtained from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan), DND-41, and P12-ICHIKAWA cells were purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. Jurkat T, DND-41, and P12-ICHIKAWA cells were cultured in 90% RPMI 1640 medium (Gibco BRL, Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS at 37 °C in a humidified incubator containing 5% CO2. Acridine orange (AO), doxycycline (Dox), 12-O-tetradecanoylphorbol-13-acetate (TPA), and ionomycin (Ion) were purchased from Sigma Aldrich (St. Louis, MO, USA).
Ionomycin ion
Manufactured by Merck Group
Sourced in United States
Ionomycin (ION) is a laboratory compound used as a calcium ionophore. It facilitates the movement of calcium ions across cell membranes, leading to an increase in intracellular calcium levels. Ionomycin is a useful tool for scientific research, particularly in the fields of cell biology and immunology.
Automatically generated - may contain errors
Lab products found in correlation
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Golgiplug, by BD (3 mentions)
Fixation permeabilization kit, by BD (2 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (2 mentions)
Percoll, by Solarbio (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Dnase 1, by Roche (2 mentions)
Brefeldin a, by BD (2 mentions)
Penicillin, by Solarbio (2 mentions)
Streptomycin, by Solarbio (2 mentions)
L glutamine, by Solarbio (2 mentions)
Dnd41, by Leibniz Institute DSMZ (1 mentions)
P12 ichikawa, by Leibniz Institute DSMZ (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Cd3ε microbead kit, by Miltenyi Biotec (1 mentions)
Anti foxp3 pe, by BD (1 mentions)
Foxp3 buffer set, by BD (1 mentions)
Anti cd4 percp, by BD (1 mentions)
Anti cd45ro fitc, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
11 protocols using ionomycin ion
1
Induction of Apoptosis in T-Cell Leukemia
2
CFSE-Based T-Cell Proliferation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD3+T cells selected with CD3ε MicroBead Kits (MiltenyiBiotec) were labeled with 5 µM CFSE (Invitrogen) for 8 min at 37°C with gentle vortex every 2 min. The labeling was terminated by adding equal volume of FCS. After washing, cells were cultured with different dose of MSCs/eGFP or MSCs/CCR7 in the presence of 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 µg/ml ionomycin (ION, Sigma) for 48 hours. Cell division, as evidenced by reduction of fluorescence intensity, was analyzed by FCM.
3
Enumeration of Th17 and Treg Cells from PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For enumeration of Th17 and Treg cells, PBMCs (1 × 106 cells) were washed and divided in two tubes. The cells in one tube were kept un-stimulated and the cells in the second tube were stimulated. Stimulation was performed with phorbol myristate acetate (PMA, 50 ng/ml, Sigma) plus ionomycin (ION, 250 ng/ml, Sigma) at 37°C and 5% CO2. Golgi-stop was added to both tubes after 1 h, and the cells were incubated for another 18 h at 37°C and 5% CO2. Then the cells were washed and stained using conjugated antibodies: anti-CD45RO-FITC (BD Pharmingen), anti-CD4-PerCP (BD Pharmingen), anti-IL-17-Alexa fluor 647 (BD Pharmingen), and anti-Foxp3-PE (BD Pharmingen) and were incubated at 4°C for 30 min. For intracellular staining of Foxp3 and IL-17 molecules, the cells were fixed and permeabilised by Foxp3 buffer set (BD, USA) before adding the conjugated Foxp3 and IL-17 antibodies. The cells were subsequently washed and resuspended in PBS containing 10% FBS. For each sample, 1 × 105 cells were acquired by FACScalibur flowcytometer. Live lymphocytes were gated on forward and side scatter, and flowcytometry analysis was carried out by FlowJo software (version 7.6.2).
4
JK-Tet-On Cell Culture Conditions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
JK-Tet-On cells were grown in a 90% RPMI-1640 medium with 10% fetal bovine serum obtained from Gibco BRL (USA) at a temperature of 37°C in a humidified 5% CO2 environment. Ribonuclease A (RNase A), acridine orange and doxycycline (Dox), 12-O-tetradecanoylphorbol-13-acetate (TPA), and ionomycin (Ion) were purchased from Sigma (USA).
5
Apoptosis and T-cell Subset Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RSC-364 cells were stained using an Annexin V/propidium iodide (PI) apoptosis detection kit (BD Biosciences, MA, USA). After 12 or 24 h of treatment, cells were harvested, washed three times with PBS, and incubated with 5 μL Annexin V-FITC and 10 μL PI for 20 min at room temperature. To measure the Th1, Th2, or Th17 cell percentages, lymphocytes were harvested and stimulated with phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) and ionomycin (Ion) (1 μg/mL) (Sigma, San Francisco, CA, USA) in the presence of GolgiPlug (BD Bioscience) for 5 h at 37°C and 5% CO2 in a cell incubator. After being surface-labeled with anti-rat CD4 PE-Cyanine5 antibody (eBioscience), lymphocytes were blocked, fixed, permeabilized using Fixation/Permeabilization kit (BD Bioscience), and stained with anti-rat IFNγ PE, IL-4 PE-Cyanine7, or IL-17A FITC antibodies (BD Bioscience). The stained apoptotic cells and Th cells were measured using a FACS Calibur cytometer, and data were analyzed using CellQuest software (Beckman Coulter, Brea, CA, USA).
6
Isolation and Stimulation of PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood of healthy volunteers and RA patients by density gradient centrifugation using Percoll (Solarbio). Human PBMCs were kept in liquid nitrogen before thawing in a 37 °C water bath. Cells were resuspended in T-cell culture medium (RPMI-1640, 10% fetal bovine serum (HyClone), 2 mM L-glutamine (Solarbio), 100 U/ml penicillin, 100 μg/ml streptomycin (both Solarbio), and 5 mM 2-mercaptoethanol (Sigma–Aldrich)). Samples were then rested overnight at 37 °C before restimulation with 50 ng ml −1 phorbol 12-myristate 13-acetate (PMA, Sigma–Aldrich) and 500 ng ml−1 ionomycin (ION, Sigma–Aldrich) in the presence of brefeldin A (BD Biosciences) for 4 h at 37 °C. Synovial tissue samples were disaggregated into single-cell suspensions as previously described (Carter et al. 2011 (link)). Murine synovial tissues were isolated from the knee joints of AIA mice and incubated with 1 mg/mL collagenase IV (Thermo Fisher Scientific) and 100 μg/ml DNase I (Roche) in RPMI in a 37 °C water bath for 45 min. Single-cell suspensions were obtained by passing cells through 70 μm cell strainers for flow cytometry staining.
7
PBMC Stimulation and IFN-γ Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell densities of fresh isolated PBMCs were adjusted. Then, 1 × 106 cells contained in 100 μl were transferred to the wells of a 96-well round-bottom microtiter plate (Costar; Fisher Scientific, Leicestershire, UK). The PBMCs were stimulated in duplicate wells with 50 μl of ASFV isolate OURT88/3, BeninΔMGF, or Benin 97/1 at a multiplicity of infection of 0.5. Mock-stimulated wells were used as negative controls, while wells stimulated with 0.2-μg/ml phorbol 12-myristate 13-acetate (PMA) and 0.2 μg/ml ionomycin (ION) (Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. Cells were incubated overnight (37°C, 5% CO2 for 14 to 15 h). Then, brefeldin A (GolgiPlug; BD Biosciences) was added (1 μl/ml) and the cells were further incubated for 6 h. Subsequently, the cells were washed twice with PBS–0.1% BSA and then incubated with 10% normal goat serum in PBS for 10 min at RT. To detect T-cell populations secreting interferon, cells were stained with MAbs (CD3, CD4α, CD8α, TCRγδ, and IFN-γ) following the protocols described above for surface markers and intracellular IFN-γ secretion.
8
Multiparametric Flow Cytometry for Immune Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were blocked with FcR antibody (anti-CD16/32, BD Biosciences, San Jose, CA, USA) and stained with conjugated antibodies in PBS containing 2% (wt/vol) BSA. Neutrophils (CD11c−SiglecF−CD11b+Ly6G+), eosinophils (CD11c−SiglecF+), B lymphocytes (CD3−CD19+), and T lymphocytes (CD3+CD19−) were detected. For intracellular cytokine analysis, cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL; Sigma), ionomycin (ION, 500 ng/mL; Sigma) together with GolgiPlug (BD Biosciences) for 5 h. Cells were collected for surface staining of CD4, CD25 (BD Biosciences), the intracellular staining of IL-4, IL-5, IL-13, IFN-γ (eBioscience), and the intranuclear staining of Ki67 (eBioscience). Viability was evaluated with an Annexin-fluorescein isothiocyanate and 7-aminoactinomycin D (7-AAD) Flow kit (BioLegend, San Diego, CA, USA). T-cells were prelabeled with carboxyfluorescein succinimidyl amino ester (CFSE, eBioscience) for detection of proliferation. All flow cytometric data were acquired from BD FACSCelesta™ and analyzed with FlowJo software (TreeStar, San Carlos, CA, USA).
9
Th1 and Th17 Cell Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Before incubation of the cells with phorbol 12-myristate 23-acetate (PMA), CD4+ T cells were purified by negative selection with CD4+ T cell isolation kit by magnetic cell sorting (Miltenyi Biotec) to avoid PMA-mediated internalization and degradation of the CD4 molecule, which would affect the identification of Th1 (CD4+IL-17-IFN-gamma+) and Th17 (CD4+IFN-gamma-IL-17+) cells [28 (link)]. Then, the cells were stimulated with 25 ng/ml PMA and 1 μg/ml of ionomycin (Ion) (Sigma-Aldrich) in the presence of 10 μg/ml of brefeldin A (BFA, protein transport inhibitor) and cultured for 4 h at 37 °C in a humidified 5 % CO2 incubator followed by the cells’ fixation and permeabilization with BD Permeabilizing Solution 2 (Becton Dickinson) according to the manufacturer’s instruction. Next, the cells were stained with phycoerythrin (PE)-labeled anti-human IL-17 (eBioscience) and fluorescein isothiocyanate (FITC)-labeled anti-human IFN-γ (Becton Dickinson) monoclonal antibodies (mAbs).
10
Intracellular Cytokine Staining for T-Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For intracellular IFN-γ and IL-2 detection, lymphocyte and TIL suspensions were stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma-Aldrich, USA) plus ionomycin (ION; 1 μg/ml; Sigma-Aldrich, USA) for 6 h. Cells were then stained with T-cell markers, further processed using a Fixation/Permeabilization kit (BD Bioscience, USA), and then labeled with a PE-conjugated IFN-γ or IL-2 mAb (BD Biosciences, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!