Single-cell suspensions of blood and skin were stained for surface or intracellular markers as previously described (Strobl et al., 2020a (link)). For intracellular cytokine staining, PBMCs were stimulated for 4 h with 1X Cell Activation Cocktail containing PMA/Ionomycin and Brefeldin A (Biolegend; 423303) in RPMI 1640 (Gibco; 52400025) according to the manufacturers’ protocol. Antibodies used are indicated in Table S1 . Stained cells were acquired on a BD Biosciences FACS Aria III using FACS Diva software or a CYTEK Aurora using SpectroFlo software. Acquired flow data were analyzed with FlowJo software v.10.6 or v.10.7 (Tree Star).
Pma ionomycin and brefeldin a
Manufactured by BioLegend
PMA/Ionomycin and Brefeldin A are laboratory reagents commonly used in cell biology research. PMA (Phorbol 12-myristate 13-acetate) and Ionomycin are typically used in combination to stimulate and activate T cells, while Brefeldin A is used to inhibit protein transport from the endoplasmic reticulum to the Golgi apparatus, leading to the accumulation of proteins within the cell. These reagents are often used in experiments involving cellular activation, cytokine production, and intracellular protein trafficking.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by Cytek (1 mentions)
Cell activation cocktail, by BioLegend (1 mentions)
Foxp3 transcription factor buffer set, by Thermo Fisher Scientific (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Zombie nir fixable viability dye, by BioLegend (1 mentions)
2 protocols using pma ionomycin and brefeldin a
1
Flow Cytometry Analysis of Cell Markers
2
ILC2 Identification by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cell suspensions were incubated with CD16/CD32, normal mouse, rat and hamster serum to block non-specific binding, then stained with fluorophore-conjugated antibodies against: CD45 (30-F11), CD11b (M1/70), CD19 (6D5), Siglec-F (S17007L), CD3 (17A2), IL7R-⍺ (A7R34). Viable cells were identified by exclusion of Zombie NIR fixable viability dye (BioLegend). For identification of ILC2, lineage-positive cells were excluded by staining for CD3 (17A2), CD4 (GK1.5), CD5 (53-7.3), CD11b (M1/70), CD11c (N418), CD19 (6D5) and NK1.1 (PK136). CD45 (30-F11), CD90.2 (30-H12), and KLRG1 (2F1) were used for positive identification. For intracellular staining, cells were re-stimulated for 4 hr with PMA/Ionomycin and Brefeldin A (Biolegend), according to manufacturer's recommendations, then fixed and permeabilized using a Foxp3/Transcription Factor buffer set (eBioscience), according to manufacturer's instructions. Cells were stained with a fluorophore-conjugated antibody against IL-13 (eBio13A). Flow cytometry was performed using a BD Fortessa X20 or CytoFLEX S (Beckman Coulter), and data were analyzed using FlowJo10.5 (TreeStar Inc.). Flow cytometry gating strategy was based on fluorescence minus one (FMO), unstained, and isotype controls.
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!