The SK-N-SH (Non-MYCN amplification) and SK-N-BE(2) cell line (MYCN amplification) (obtained from the Shanghai Institute of Biological Cell Research, Chinese Academy of Sciences) was cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS; Gibco) [22 (link), 23 ]. Additionally, the THP-1 cell line (also obtained from the Shanghai Institute of Biological Cell Research, Chinese Academy of Sciences) was cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/ml penicillin–streptomycin. HUEVC Human vascular endothelial cells were isolated by collagenase digestion of the human umbilical cord veins obtained from Children's Hospital Affiliated with Chongqing Medical University with the informed consents signed by donors. Human vascular endothelial cells were cultured in Endothelial Cell Medium (Lonza). 0.99 μm BKM 120 for SK-N-SH, 1.2 μm BKM 120 or SK-N-BE(2) (one of a PI3K inhibitor) (MCE)was used for the PI3K-AKT experiment [24 (link)]. For THP-1 cells, 100 ng/ml of PMA (Phorbol 12-myristate 13-acetate) (Multi-Science) was added, and recombinant human CXCL14 (MCE) was used for the experiment in vitro [25 (link)].
Phorbol myristate acetate (pma)
Manufactured by MultiSciences Biotech
Sourced in China
PMA is a versatile laboratory equipment designed for the analysis and processing of biological samples. It serves as a crucial tool for researchers and scientists working in various fields, including microbiology, molecular biology, and biochemistry. The core function of PMA is to provide a controlled environment for the manipulation, incubation, and observation of samples, enabling accurate and reliable data collection.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by MultiSciences Biotech (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Endothelial cell medium (ecm), by Lonza (1 mentions)
Phorbol 12 myristate 13 acetate, by MultiSciences Biotech (1 mentions)
Ionomycin, by MedChemExpress (1 mentions)
Phorbol myristate acetate (pma), by MedChemExpress (1 mentions)
Gsk 5498a, by MedChemExpress (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Il 17a pe, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
1640 medium, by Thermo Fisher Scientific (1 mentions)
Lonomycin, by MultiSciences Biotech (1 mentions)
Facscalibur flow cytometer, by Beckman Coulter (1 mentions)
Monensin, by MultiSciences Biotech (1 mentions)
Penicillin streptomycin, by Solarbio (1 mentions)
Macrophage colony stimulating factor m csf, by Thermo Fisher Scientific (1 mentions)
Hepes, by Solarbio (1 mentions)
Cd4 pacific blue, by Thermo Fisher Scientific (1 mentions)
12 protocols using phorbol myristate acetate (pma)
1
Cell Culture and Treatment Protocols
2
Modulation of CD4+ T Cell Activation by UC-MSC-EVs
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The CD4+ T cells used in in vitro experiments were prepared from the spleen of C57BL/6 mice by positive selection using a mouse CD4+ T cells MicroBeads UltraPure kit (Miltenyi Biotec, Germany) according to the manufacturer's protocol. Flow cytometry was used to determine the purity of CD4+ T cells, which was more than 95%.
Group design: To explore the mechanism that how UC‐MSC‐EVs regulate CD154 expression on CD4+ T cells, in vitro model was also established, which was randomly divided into six groups, including: normal group, CD4+ T cells were cultured in the typical medium without any intervention; Control group, CD4+ T cells were stimulated by PMA (MultiSciences, Hangzhou, China) and ionomycin (MultiSciences) without any other treatment; UC‐MSC‐CM group, CD4+ T cells were incubated with PMA and ionomycin and treated with UC‐MSC‐CM; UC‐MSC‐ECM group, UC‐MSC‐CM was replaced by UC‐MSC‐ECM; UC‐MSC‐EV group, CD4+ T cells were subjected to PMA and ionomycin stimulation as well as treated with UC‐MSC‐EVs; GSK‐5498A group, CD4+ T cells were treated with GSK‐5498A (calcium channel blocker, 1 × 10−6m , MedChem Express, Monmouth Junction, NJ, USA)[33 ]; UC‐MSC‐EVshCCT2 group, CD4+ T cells were treated with UC‐MSC‐EVsshCCT2 and incubated in the medium containing PMA and ionomycin.
Group design: To explore the mechanism that how UC‐MSC‐EVs regulate CD154 expression on CD4+ T cells, in vitro model was also established, which was randomly divided into six groups, including: normal group, CD4+ T cells were cultured in the typical medium without any intervention; Control group, CD4+ T cells were stimulated by PMA (MultiSciences, Hangzhou, China) and ionomycin (MultiSciences) without any other treatment; UC‐MSC‐CM group, CD4+ T cells were incubated with PMA and ionomycin and treated with UC‐MSC‐CM; UC‐MSC‐ECM group, UC‐MSC‐CM was replaced by UC‐MSC‐ECM; UC‐MSC‐EV group, CD4+ T cells were subjected to PMA and ionomycin stimulation as well as treated with UC‐MSC‐EVs; GSK‐5498A group, CD4+ T cells were treated with GSK‐5498A (calcium channel blocker, 1 × 10−6
3
Intracellular Cytokine Staining Assay
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Cells were stained with FITC-CD4, APC-IL-4 and PE-IL-17A (all from eBioscience, San Diego, CA, USA). For intracellular IL-17A staining, single-cell suspensions from the lung were stimulated for five hours with 10 ng/ml PMA (Phorbol myristate acetate, Multi Sciences, china), then 500 ng/ml ionomycin (Multi Sciences, china), and 10 μg/ml BFA (brefeldin A, Multi Sciences, china) was added during the final four hours of incubation. After stimulation, cells were harvested, followed by surface and intracellular staining according to the manufacturer’s instructions (eBioscience, San Diego, US). Stained leukocytes were washed and fixed in 2% paraformaldehyde and analyzed by flow cytometry using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA, US). Positive staining thresholds were determined from appropriate isotype control staining.
4
Maintaining and Differentiating Cell Lines for RSV Studies
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HEp-2 cells and RSV A strain Long were maintained as previously described (32 (link)). HEp-2 cells were utilized to amplify RSV (34 (link)). Human alveolar epithelial cells (A549), human bronchial epithelial cells (BEAS-2B), and human monocytic leukemia cell line (THP-1) were maintained in our laboratory and grown in RPMI 1640 supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Gibco) and antibiotics (penicillin and streptomycin) (Solarbio) in a humidified 5% CO2 atmosphere at 37°C. Monocytes (THP-1) were treated with phorbol 12-myristate 13-acetate (PMA, 15 ng/ml; Multiscience) and were differentiated into macrophages MΦ-THP-1 after 12 h.
5
Polarization of THP-1 Macrophages
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THP‐1 macrophages were induced with 50 ng/mL PMA (MultiSciences, China) for 48 h to polarize the cells into M0 macrophages, and then recombinant human CCL5 protein (Genscript, China) was applied at a concentration of 20 ng/mL to induce macrophage polarization for 72 h. Later, according to the protocol, the cells were fixed with paraformaldehyde, followed by incubation with 5% BSA for 1 h. Then, flow cytometry antibodies specific for M1 (CD16) and M2 (CD206) macrophage markers (BD Pharmingen) were added and cells incubated at 4℃ for 1.5 h. The phenotypes of polarized cells were detected using flow cytometry.
6
Flow Cytometric Analysis of Th17 and Treg Cells
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The proportions of Th17 and Treg cells were detected using flow cytometry. In brief, lymphocytes were isolated from the spleens of mice and washed twice by phosphate-buffered saline (PBS) solution. Subsequently, the cells were transferred to test tubes and incubated with surface antibody of FITC-labeled CD4 (rat anti-mouse, BD, 553046, USA) and APC-labeled CD25 (rat anti-mouse, BD, 557192, USA). After being washed again by PBS, 1 ml of 1640 medium (Gibco, USA) containing phorbol 12-myristate 13-acetate (PMA, MultiSciences, China), lonomycin (MultiSciences, China), and monensin (MultiSciences, China) was added in each tube, incubated at 37°C for 4 h, and washed again. Next, cells were fixed and permeated successively, followed by incubating with intracellular antibody, including PE-labeled IL-17A (rat anti-mouse, BD, 559502, USA), PE-labeled retinoic acid−related orphan receptor γt (RORγt, rat anti-mouse, ebioscience, 12-6981-60, USA), and PE-labeled FoxP3 (rat anti-mouse, BD, 563101, USA), at a temperature of 4°C for 60 min. After being washed again and analyzed by a FACSCalibur flow cytometer (Beckman Coulter, USA), the resulting data were processed by FlowJo software (Tree Star, Ashland).
7
Macrophage Differentiation and Culture
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The human monocytic leukemia cell line THP-1 (resource center of Peking Union Medical College Hospital, China) and HEK 293T cells (Shanghai stem cell bank) were cultured in RPMI 1640 (Gibco) containing penicillin-streptomycin (100mg/mL for each drug, Solarbio), 10mM HEPES (Solarbio), 0.05mM 2-mercaptoethanol, and 10% fetal bovine serum (Gibco) at 37°C with 5% CO2 in a humidified atmosphere. Before each test, monocytes (THP-1) were differentiated into macrophages (MΦ-THP-1) after treated with phorbol 12-myristate 13-acetate (PMA, 15ng/mL, multiscience) for 48h. Mouse BMDMs were isolated and cultured using standard protocols [46 (link), 47 (link)]. Bone marrow cells from BALB/c mice were collected by flushing the femurs from six-to eight-week-old mice with phosphate buffered saline (PBS) and then cultured in RPMI 1640 (Gibco) containing 10% fetal bovine serum (BI) and macrophage-colony-stimulating factor (M-CSF) (Peprotech) at 37°C with 5% CO2 for 7 days. After 7 days of culturing, non-adherent cells were eliminated. Adherent cells, which were highly enriched in BMDM, were recovered for studies. All the animal procedures were vetted and approved by the Hebei Medical University Ethical Committee for Animal Experimentation.
8
Cell Viability Assay for PMA Treatment
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Using a Cell Counting Kit-8 (CCK8) (Share Bio, Shanghai, China) assay, cell viability curves were established for the dose- and time-dependent effects of phorbol 12-myristate 13-acetate (PMA) (Multi Sciences, Hangzhou, China) on THP-1 cells.
THP-1 cells were inoculated in a 96-well plate, at a density of 1 × 104 cells/well and treated with different concentrations of PMA (0, 20, 50, 100, and 200 ng/ml)/well for 24 h to determine the dose effect. According to CCK-8 kit instructions, the absorbance of each well was measured according to the manufacturer's instructions. Cell viability was calculated using the following formula: cell viability (%) = (Abs 450 nm of administered cells – Abs blank group)/(Abs negative group – Abs blank group) × 100.
To determine the time effect of PMA, THP-1 cells were seeded in 96-well plates and the optimal PMA concentrations were determined by the dose-effect curve. Cell viability was measured at different time points (0, 12, 24, and 48 h) using the CCK-8 assay, and cell viability was calculated to obtain a time-effect curve.
THP-1 cells were inoculated in a 96-well plate, at a density of 1 × 104 cells/well and treated with different concentrations of PMA (0, 20, 50, 100, and 200 ng/ml)/well for 24 h to determine the dose effect. According to CCK-8 kit instructions, the absorbance of each well was measured according to the manufacturer's instructions. Cell viability was calculated using the following formula: cell viability (%) = (Abs 450 nm of administered cells – Abs blank group)/(Abs negative group – Abs blank group) × 100.
To determine the time effect of PMA, THP-1 cells were seeded in 96-well plates and the optimal PMA concentrations were determined by the dose-effect curve. Cell viability was measured at different time points (0, 12, 24, and 48 h) using the CCK-8 assay, and cell viability was calculated to obtain a time-effect curve.
9
Cytokine Production Assay in PBMCs
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The PBMCs were incubated for four hours with 50 ng/mL of phorbol 12-myristate 13-acetate (PMA; MultiSciences Biotech, Hangzhou, China), 1 μg/mL of ionomycin (MultiSciences Biotech), and 500 ng/mL of monensin (eBioscience, San Diego, CA, USA) in a 6-well plate. The cells were initially stained for Pacific Blue-CD4 (eBioscience) on the surface. Then, these were fixed and permeabilized with IC Fixation/Permeabilization buffer (eBioscience), washed, and intracellularly stained with PE-Cy7-IL-17 (BioLegend, San Diego, CA, USA) and FITC-IL-22 (BioLegend). Finally, these cells were collected and analyzed using BD FACSVerse (BD Biosciences, NJ, USA). The flow cytometric data analysis was performed using the FlowJo software, version 10.0 (Tree Star Inc., San Carlos, CA, USA).
10
Differentiation of THP-1 Cells into Macrophages
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The human monocytic leukemia cell line (THP-1) came from the American Type Culture Collection (ATCC) and was cultured in Roswell Park Memorial Institute (RPMI) medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Gibco) at 37°C in 5% CO2. For the experiments, the cells were seeded on a 12-well plate or a 6-well plate. Each well contained 106 and 2 × 106 cells, respectively. The cells were cultured for 36 h in the presence of a phorbol ester (PMA; Multi Sciences, Hangzhou, China; 100 nM) to induce differentiation into macrophages (Lyu et al., 2022 (link); Shah et al., 2022 (link)). The cells were washed and incubated for an additional 24 h in a fresh medium without PMA until they were used in experiments. Before infection, approximately 70%–80% of the cells had already adhered and had some expanded pseudopodia, which were then visualized under an optical microscope.
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