PBMCs collected in clinics were kept in cell culture medium (RPMI-1640, 10% fetal bovine serum (FBS; GIBCO), and 1 × l-glutamine (GIBCO) and 1 × penicillin streptomycin (GIBCO)) supplemented with 5U ml−1 benzonase (Sigma–Aldrich) and frozen in liquid nitrogen until experimental analysis. Then, for spectral flow analysis, cells were thawed using Cryo thaw devices (Medax). Briefly, cells were resuspended in cell culture medium supplemented with 2U ml−1 benzonase by centrifugation (300 r.c.f.; 7 min; 24°C). Cell count was calculated using an automated cell counter (Bio-Rad). Due to the resulting cell count, cells were used for all panels or surface panel only. Subsequent procedure including short-term reactivation of cryopreserved PBMCs and cytometry analysis were performed as described previously (Galli et al., 2019 (link); Hartmann et al., 2016 (link)). Briefly, 2 million (mio) cells were directly stained for cytometry analysis (surface panel), while 1 mio cells were restimulated with 50 ng ml−1 phorbol 12-myristate 13-acetate (Sigma–Aldrich) and 500 ng ml−1 ionomycin (Sigma–Aldrich) in the presence of 1 × Brefeldin A and 1x Monensin (both BD Biosciences) for 5 h at 37°C or in case of R848 stimulation, 2.5 mio cells using 2μg ml−1 R848 (Invivogen) in the presence of 1 × Brefeldin A and 1x Monensin (both BD Biosciences) for 8 h at 37°C.
Monensin
Manufactured by BD
Sourced in United States, China, Italy
Monensin is a laboratory product that functions as an ionophore, a compound that facilitates the transport of ions across cell membranes. It is commonly used in cell culture and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by BD (120 mentions)
Ionomycin, by Merck Group (81 mentions)
Phorbol myristate acetate (pma), by Merck Group (39 mentions)
Brefeldin a, by Merck Group (28 mentions)
Cytofix cytoperm kit, by BD (26 mentions)
Cytofix cytoperm, by BD (22 mentions)
Perm wash buffer, by BD (15 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (14 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (12 mentions)
Lsrfortessa, by BD (11 mentions)
Anti cd28, by BD (10 mentions)
Golgistop, by BD (10 mentions)
Golgiplug, by BD (9 mentions)
Lsrii flow cytometer, by BD (9 mentions)
Facscalibur, by BD (8 mentions)
Anti cd49d, by BD (7 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (7 mentions)
Lsrfortessa flow cytometer, by BD (7 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (7 mentions)
Fortessa, by BD (7 mentions)
326 protocols using monensin
1
Cryopreserved PBMC Reactivation and Analysis
2
HIV Gag Peptide Stimulation Assay
3
HIV Gag Peptide Stimulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were incubated for 6 hours with an HIV Gag peptide pool at a concentration of 0.5μg/ml/peptide in the presence of 2.5 μg/ml brefeldin A (BD GolgiPlug) and (0.3μl/ml) monensin (BD GolgiStop). For delayed ICS assays, 2.5 μg/ml brefeldin A (BD GolgiPlug) and (0.3μl/ml) monensin (BD GolgiStop) were added 9h after stimulation with the Gag peptide pool and cells were cultured for 12h. Unstimulated cells were used as negative control and SEB (0.5) μg/ml as positive control. Cells were then stained with viability dye (20 min at 4 C°), surface markers (20 min at 4 C°), fixed with Fixation Solution (eBioscience), and stained for intracellular proteins in permeabilization 1X buffer (eBioscience) (30 min at 4 C°) as described in Supplementary Tables 15 and 16 . Cells were acquired on an LSR II (BD Biosciences).
4
Intracellular Cytokine and Transcription Factor Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For intracellular detection of in vitro cytokine and transcription factor production, cells were plated at a density of 2 × 106 ml in RPMI1640 (Gibco) containing 10% FBS, L-glutamine, and 2-mercaptoethanol. Cells were stimulated for 4-6 hours using either 1 μg/ml each, plate-bound anti-CD3ε f(ab′)2 (2C11, BioXCell), and anti-CD28 (37.51, BioXCell) in the presence of GolgiStop (3 mM monensin, BD), or Cell Stimulation Cocktail plus protein transport inhibitor (50 ng/ml phorbol 12-myristate 13-acetate (PMA), 1.34 mM ionomycin, 5.3 mM brefeldin A, 1 mM monensin) according to manufacturer guidelines (eBio). After surface staining, cells were fixed and permeabilized according to manufacturer guidelines (BD). Subsequent intracellular staining was performed with anti-TNFα (BD, MP6-XT22). Foxp3 (Fjk-16s, eBio) was detected in lymphocytes ex vivo following fixation and permeabilization according to manufacturer guidelines (eBio).
5
Activating and Analyzing PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For phorbol 12-myristate 13-acetate (PMA)/ionomycin-based stimulation, PBMCs were cultured for 4 hr in RF10 complete media (RPMI-1640 (Invitrogen, Life Technologies) supplemented with 10% (v/v) FBS (JRH Biosciences), 2% (v/v) Penicillin (100 U/ml), Streptomycin (100 μg/ml), Glutamax (2 mM), sodium pyruvate (1 mM), nonessential amino acids (0.1 mM), HEPES buffer (15 mM), pH7.2-7.5 (all from Invitrogen, Life Technologies) and 2-mercaptoethanol (50 μM, Sigma)) in the presence of 10ng/ml PMA, 1 μg/ml ionomycin and 1/500 Monensin (BD Biosciences). Cells were then harvested and stained as above.
For E. coli stimulation assays, PBMCs were co-cultured for 20 hr in human RF10 complete media in the presence of PFA-fixed DH5α E. coli (MOI 0.25 was determined as optimum), anti-CD107a mAb and anti-MR1 blocking mAb (clone 26.5, produced in-house). In the final 6 hr of culture, Monensin (1:1500) and Brefeldin A (1:1000; both BD Biosciences) were added to the cultures. Cells were then harvested and stained as above. For Len/E. coli stimulation assays, no Monensin was added.
For E. coli stimulation assays, PBMCs were co-cultured for 20 hr in human RF10 complete media in the presence of PFA-fixed DH5α E. coli (MOI 0.25 was determined as optimum), anti-CD107a mAb and anti-MR1 blocking mAb (clone 26.5, produced in-house). In the final 6 hr of culture, Monensin (1:1500) and Brefeldin A (1:1000; both BD Biosciences) were added to the cultures. Cells were then harvested and stained as above. For Len/E. coli stimulation assays, no Monensin was added.
6
Intracellular IL-17 Quantification in PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure intracellular IL-17, PBMCs after culturing with PPD were stimulated with PMA (50 ng/ml) plus ionomycin (1 μg/ml) and monensin (3 μM monensin) (BD, USA) for 4 hr in 1 ml RPMI 1640 media containing 10% fetal calf serum (FCS). For surface staining, FITC anti-CD4 was added. Cells were washed with PBS containing 2% FCS and were fixed/permeabilized with %2 fixation/permeabilization buffer and stained with the anti-human IL-17PE antibody (BD, USA). After staining, cells were analyzed using CellQuest flowcytometer (BD. USA).
7
Intracellular Cytokine Profiling of NK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NK cells were subjected to intracellular cytokine-staining assay following 1 h incubation with monensin (2 mM, BD) or to evaluate the production of IFNγ, after an overnight stimulation with PMA (10 ng/ml, Sigma-Aldrich, Milan, Italy) and Ionomycin (500 ng/ml, Sigma-Aldrich) plus monensin (2 mM, BD). Briefly, following staining with anti-human mAbs CD3-PerCP, CD56-APC, and CD16-FITC (Miltenyi Biotec), cells were permeabilized and fixed using the Cytofix/Cytoperm fixation kit (BD), according to the manufacturer's instructions and finally stained with different anticytokine PE-conjugated mAbs (VEGF, SDF-1, perforin, osteopontin, IL-8, or IFNγ, all from Miltenyi Biotec).
8
T Cell Activation Assays for Peptide Screening
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In the 18mer and 13mer peptide screening assays, bulk cultured T cells were incubated with peptide at 5μM in “RP-5” at 37°C for 5h in the presence of monensin (Becton Dickinson). In assays assessing the restricting HLA alleles, BLCLs were pulsed with peptide of interest at 5μM for 1h, washed extensively and then co-cultured with bulk cultured T cells at a ratio of 1:10 for 5h in the presence of monensin. The cells were harvested, stained and analyzed by FACS as we described before [20 (link)]. In the antibody-blocking assay, APC were incubated with 10 μl of anti-HLA class II antibody supernatant for 30 min before addition of peptide and monensin. T cell activation was subsequently measured by ICS.[36 (link)]
9
Detecting NKT-like Cell Functions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The functions of NKT-like cells in PBMCs were detected after coincubation with the K562 cell line or PMA/ionomycin. Because NKT-like cells share some features with NK cells, they can recognize HLA class I-negative cell line K562 and their functions can be detected through coculture with K562 [10 , 11 (link)]. PBMCs were coincubated for six hours with K562 cell line at an E : T ratio of 5 : 1. Meanwhile, PBMCs were stimulated with PMA (Sigma, Cat. No. P-8139, USA) and ionomycin (Sigma, Cat. No. I-0634, USA) in final concentrations of 50 ng/mL and 1 μM, respectively. PE-conjugated anti-CD107a (BD Biosciences, USA) and monensin (Becton-Dickinson, USA) were added to all incubated samples. Then cells were stained with both Percp-conjugated anti-CD3 and PE-cy7-conjugated anti-CD56 (BD Biosciences, USA). The cells were made permeable using Perm/Wash (Becton-Dickinson, USA) for 10 minutes, stained with FITC-conjugated anti-IFN-γ (BD Biosciences, USA) for 30 minutes at 4°C, washed, and then fixed in 1% formaldehyde. NKT-like cell populations were defined by dual-positive expressions of CD3 and CD56 molecules. The frequency of IFN-γ and CD107a expression in NKT-like cells were quantified by multicolor flow cytometry (Figure 1 ).
10
PBMC Stimulation and Cytokine Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK) from peripheral venous blood sample (10mL) collected from the study participants. Isolated PBMCs(106 cells/mL) were stimulated using a commercial leukocyte activation cocktail (10μg/mL; Becton Dickinson, USA) in the presence of a protein transport inhibitor containing monensin(3.5μg/ml; Becton Dickinson, USA)while being maintained in RPMI 1640 medium at 37°C in a humidified incubator with 95% air and 5% carbon dioxide for 4 hours. Parallel cultures in which the activation cocktail was not added served as negative control.
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!