FLP293T derived stable cell lines were cultured in DMEM supplemented with 10% dialyzed fetal bovine serum (FBS). Wildtype and Crbn−/− Jurkat cells were cultured in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin in a 37°C incubator with 5% CO2. CD4+ CD25+ regulatory T cells were purified from spleens from wildtype or CrbnI391V/I391V mice using the EasySep Mouse CD4+ CD25+ Regulatory T cell Isolation Kit (StemCell Technologies). T cells were cultured in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% non-essential amino acids, 1% sodium pyruvate, 2 mM GlutaMax, 10 mM HEPES, and 50 μM 2-mercaptoethanol, as well as 5 ng/ml recombinant murine IL-2 (Biolegend) and 20 ng/ml recombinant murine IL-4 (Biolegend). Human regulatory T cells were isolated from peripheral blood mononuclear cells using the EasySep Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (StemCell Technologies) and expanded for 12–14 days in the presence of 500 U/ml human IL-2, Immunocult CD3/CD28 T cell activator (StemCell Technologies), and compound in ImmunoCult-XF T Cell Expansion Medium (StemCell Technologies). Cells were stimulated with 1X PMA/ionomycin (Biolegend) for 1h followed by the addition of brefeldin A (Biolegend) for another 3h.
Pma ionomycin
Manufactured by BioLegend
Sourced in United States
PMA/ionomycin is a cell stimulation reagent used to activate T cells and other immune cells. It consists of phorbol 12-myristate 13-acetate (PMA) and ionomycin, a calcium ionophore. Together, these compounds stimulate multiple signaling pathways, leading to the activation and proliferation of T cells and other immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by BioLegend (8 mentions)
Golgiplug, by BD (6 mentions)
Cytofix cytoperm kit, by BD (5 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
Recombinant murine il 2, by BioLegend (2 mentions)
Recombinant murine il 4, by BioLegend (2 mentions)
Easysep human cd4 cd127lowcd25 regulatory t cell isolation kit, by STEMCELL (2 mentions)
Human il 2, by STEMCELL (2 mentions)
Immunocult cd3 cd28 t cell activator, by STEMCELL (2 mentions)
Easysep mouse cd4 cd25 regulatory t cell isolation kit, by STEMCELL (2 mentions)
Immunocult xf t cell expansion medium, by STEMCELL (2 mentions)
Pd l1, by BioLegend (2 mentions)
Ifn γ, by BioLegend (2 mentions)
Hybrimax grade, by Merck Group (2 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (2 mentions)
Cd69 pe cy7, by BioLegend (2 mentions)
Live dead apc cy7, by BioLegend (2 mentions)
Cd94 bv421, by BioLegend (2 mentions)
Facs symphony a5, by BD (2 mentions)
Ly6g apc, by BioLegend (2 mentions)
32 protocols using pma ionomycin
1
Crbn Knockout Regulatory T Cell Culture
2
Eosinophil Peroxidase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow-derived eosinophils were incubated with LPS (Sigma-Aldrich), IL-33, and Phorbol 12-myristate 13-acetate (PMA)-ionomycin (Biolegend) for 12 h at the indicated concentration. The EPO activity was measured by the spectrophotometric method (31 (link)). Briefly, 100 µl of culture supernatant from each sample was placed in a 96-well plate, and 100 µl of substrate solution containing 0.1 µM o-phenylenediamine-dihydrochloride, 0.1% Triton X-100, and 1 µM hydrogen peroxide (Sigma-Aldrich) was added in each well. After incubation for 30 min at 37°C, the enzymatic reaction was stopped by adding 50 µl of 4 M sulfuric acid. Absorbance was measured at 492 nm using a Multiscan JX system (Thermo Fisher Scientific).
3
Fecal Microbiome Impacts T Cell Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To test whether faecal microorganisms from C57BL/6J mice fed with different methionine diets have distinct abilities to activate T cells, whole blood collected from naive C57BL/6J mice fed with chow diet was treated with 1× RBC Lysis Buffer (Invitrogen, 00-4300-54) for 15 min to lyse red blood cells. The resulting PBMCs were cultured in RPMI 1640 + 10% FBS in a 12-well plate at 1 × 106 cells per well overnight. The next day, five fresh faecal pellets were collected from C57BL/6J mice fed with either control diet or MR diet, washed with 1× PBS three times. The washed faecal microorganisms were then added to the cultured PBMCs with a 1:1,000 ratio of bacteria:cells and incubated for an additional 6 or 12 h. The same amount of PBMCs incubated with CD3/CD28 beads and PMA/ionomycin (BioLegend, 423301) were used as a positive control. PBMCs without any treatment were used as a negative control. The fraction of IFN-γ+CD8+ T cells was analysed by flow cytometry.
4
Evaluating NK Cell-Mediated Cytotoxicity Against Glioblastoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The GBM cells U87, U118, U343, and pGBM (4 × 104 cells/wells) with or without treatment with Bev and Iri were used as target cells. The NK-HD and NK-GBM cells were co-cultured with target cells at a 1:3 (target:effector) ratio in 96-well plates (SPL, Gyeonggi-do, Korea) under a 5% CO2 atmosphere at 37°C for 5 h. Subsequently, the supernatants were collected to determine the IFN-γ concentration by using the OptEIA ELISA kit (BD Bioscience) as per the manufacturer’s instructions. NK alone and NK treated with dimethyl sulfoxide (DMSO) were used as the negative control and NK treated with PMA/Ionomycin (Biolegend, 423302) was used as the positive control for released IFN-γ.
5
Cytokine Stimulation and NK Cell Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were stimulated ex vivo with PMA/Ionomycin (1:500, BioLegend) IL-12 (10ng/mL), IL-18 (10ng/mL), IL-23 (10 ng/ml) or IL-1β and IL-23 (10 ng/ml) for 4 hours as indicated. For intracellular cytokine staining, brefeldin A was present for the last 3.5 hours of stimulation. For Yac-1:NK co-culture experiments, NK cells were cultured with Yac-1 at a target:effector ratio of 20:1 for 6 hours at 37C. For NK1.1 crosslinking studies, 2μg of anti-NK1.1 (PK136) was added to 12-well plates and incubated at 4C overnight. 5×106 total splenocytes were added to each well and incubated for a total of 6 hours. Cells were stained with anti-CD107a for 1 hour and then cultured with brefeldin A and monensin for 5 hours.
6
Multiparametric T Cell Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were stained with CD3 (BioLegend, clone:17A2), CD4 (BioLegend, clone: GK1.5), CD8 (BioLegend, clone:53-6.7), GZMB (Biolegend, clone: QA16A02), IFN-γ (Biolegend, clone: XMG1.2), Ki-67 (BioLegend, clone: 16A8), PD-1(Biolegend, clone:29F.1A12), PD-L1 (BioLegend, clone:10F.9G2), CD44 (Biolegend, clone: IM7), CD62L (BioLegend, clone: MEL-14), CD25(BioLegend, clone: 3C7), CD69(BioLegend, H1.2F3), BCL2 (Cell Signaling Technology, clone: 124), PRF(BioLegend, clone: S16009A). For intracellular staining, cells were given PMA/ionomycin (BioLegend, Cat. No. 423303) re-stimulation for 4–5 h, fixed, permeabilized, and then stained. FACS analysis was performed on a BD FACS Aria III flow cytometer and analyzed by FlowJo V.10 software. BD FACS Aria III flow cytometer was used to perform FACS analysis and data analysis was performed in FlowJo V.10 software.
7
Intracellular Cytokine Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cell suspensions from spleens were generated by passage of tissue through a 100 micron strainer. Single cell suspensions from livers were generated by passage through a 100 micron strainer and leukocytes enriched via a percoll (Sigma) gradient. Red blood cells were lysed with ACK lysis buffer (Gibco). For intracellular cytokine staining, cells were plated at 1–3 × 106 cells per well. Cells were stimulated in RPMI media (Gibco) supplemented with 10% fetal bovine serum (Gibco), penicillin, streptomycin, brefeldin A (Biolegend), monensin (Biolegend) and PE-conjugated CD107a (0.5 μg/mL, Biolegend). Cells were stimulated with DMSO vehicle (Hybrimax grade, Sigma), CCHFV peptides at 1 μg/mL each peptide or PMA/ionomycin (Biolegend) for 6 h at 37 °C. Cells were then processed for flow cytometry.
8
Activation and Analysis of NK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An isolation kit (Stemcell, CAN) was used to isolate the NK cells, which were activated by Phorbol 12-myristate 13-acetate (PMA)/ionomycin (BioLegend, USA) and protein transport inhibitor GolgiStop (BD Biosciences) in 96-well U-bottomed plates with 5% CO2 at 37 °C for 6 h. First, the NK cells were collected, and Fixable viability stain 620 (BD Pharmingen, USA) was added to exclude dead cells. Antibodies for cell surface staining included anti-CD3-APC-CY7, anti-CD56-PE-Cy7, anti-CD38-PE, anti-CD39-FITC (BioLegend, USA) and were incubated for 20 mins. Next, the cells were fixed and washed by Fixation/Permeabilization solution (BD Biosciences, USA) and intracellularly labeled by anti-IL-10-APC and anti-TGF-β-BV421 (BD Biosciences, USA) at 4 °C for 20 mins. Finally, the cells were measured by LSR II Fortessa cytometer (BD Biosciences, USA).
9
Multiparametric Immune Profiling of T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were stained with CD3 (BioLegend, clone: HIT3a), CD8 (BioLegend, clone: SK1), CD45RO (BioLegend, clone: UCHL1), T-bet (BioLegend, clone: 4B10), Eomes (R&D Systems, clone: 644730), NFATC1 (BioLegend, clone: 7A6), PD-1 (BioLegend, clone: EH12.2H7), TIM-3 (BioLegend, clone: F38-2E2), CTLA4 (BioLegend, clone: L3D10), LAG3 (BioLegend, clone: 11C3C65), Bcl-XL (Abcam, clone:7B2.5), FABP5 (R&D Systems, clone: 311215)，CPT1α (Proteintech, 15184-1-AP), CD137 (BioLegend, clone: 4B4-1), mito Tracker Red CMXROS (Invitrogen, M7512), TMRM (Invitrogen, M20036), LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, L3224), CD27 (BioLegend, clone: M-T271), CD127 (eBioscience, clone: eBioRDR5), IFNγ (BD Pharmingen, clone: B27), TCF7 (BioLegend, clone: 7F11A10), IRF4 (BioLegend, clone: IRF4.3E4), Blimp1 (BD Pharmingen, clone: 6D3), BCL2 (Cell Signaling Technology, clone: 124), Ki67 (Abcam, clone: B126.1), PPARγ (Abcam, clone: EPR18516), Annexin V (BD Pharmingen, 556547), CFSE (BD Pharmingen, 565082), KLRG1 (BioLegend, clone: 2F1/KLRG1), as well as BODIPY 493/503 (Invitrogen, D3922) and BODIPY FL C16 (Invitrogen, D3821). For intracellular staining, cells were given PMA/ionomycin (BioLegend, 423303) re-stimulation and then intracellular staining was performed as previously described.6 (link) FACS analysis was performed on a BD FACS Aria II flow cytometer and analyzed by FlowJo V.6 software.
10
Hamster PBMC Isolation and Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hamster peripheral blood mononuclear cells (PBMCs) were isolated from ethylene diamine tetraceticacid (EDTA) whole blood by overlay on a Histopaque®-1077 density cushion and separated according to manufacturer’s instructions. Tissues were processed into single cell suspensions as described previously (44 (link)). Cells were stimulated for 6 hours with media alone, cell stimulation cocktail (containing PMA-Ionomycin, Biolegend), 1μg/ml SARS-CoV-2 S peptide pool (IDT), or Lassa virus (LASV) GPC peptide pool (IDT) together with 5μg/ml Brefeldin A (Biolegend). Following surface staining with Live/Dead-APC/Cy7, CD4-FITC, CD8-Alexa700, CD94-BV421 and CD69-PeCy7, B220-BV605, CD11b-PerCPCy5.5, and Ly6G-APC (all Biolegend) cells were fixed with 4% paraformaldehyde (PFA). Sample acquisition was performed on a FACSSymphony-A5 (BD), and data analyzed in FlowJo V10. Cell populations were identified by initially gating on Live/Dead negative, doublet negative (SSC-H vs SSC-A). Activation positive responses are presented after subtraction of the background responses detected in the LASV GPC peptide pool-stimulated samples.
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!