In order to evaluate the impact of an additional treatment with ethidium monoazide (EMA) or propidium monoazide (PMA) during DNA extraction for a selective removal of unprotected DNA, 100 μM EMA or PMA (Biotium, VWR, Germany) were applied as described previously (Wagner et al., 2008 (link)). After EMA or PMA addition the samples were vortexed for 5 s and incubated for 10 min in the dark in order to allow EMA and PMA, respectively, to penetrate the cells with damaged cell walls, followed by a 30 min light incubation (650 W halogen) for the activation of EMA and PMA, respectively, by binding to DNA. In order to avoid the samples getting heated, light activation was done on ice. Samples were mixed very gently in 5 min intervals. Following to EMA/PMA treatment the DNA extraction procedure was conducted according to the respective protocol starting with the beat-beating step.
Phorbol myristate acetate (pma)
Manufactured by Avantor
Sourced in Germany
The PMA is a lab equipment product that serves as a peristaltic pump. It is designed to precisely control the flow of fluids in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Avantor (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
96 well tissue culture plate, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
Perm wash, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Dnase, by Merck Group (1 mentions)
Fortessa, by BD (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Foxp3 fix perm buffer, by BioLegend (1 mentions)
Foxp3 perm buffer, by BioLegend (1 mentions)
Ifn γ pe xmg1.2, by BioLegend (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Biomek fx, by Beckman Coulter (1 mentions)
Imagexpress micro confocal high content imaging system, by Molecular Devices (1 mentions)
6 protocols using phorbol myristate acetate (pma)
1
EMA/PMA-Assisted DNA Extraction Protocol
2
Differentiation of THP-1 Monocytes to Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
THP-1 cells (kind
gift from Prof. David Hume) were maintained in RPMI-1640 medium (Thermo
Fisher) supplemented with 10% heat inactivated fetal bovine serum
(FBS, Life Technologies) and GlutaMax (Thermo Fisher) at 37 °C
in a 5% CO2 environment. Preparation of THP-1 cells prior
to infection, THP-1 monocytes were differentiated into macrophages
by treatment with 50 nM phorbol 12-myristate 13-acetate (PMA, VWR)
for 3 days at a density of 5 × 104 cells per well
in a 96-well tissue culture plate (Thermo Fisher, Nunclon Delta).
The media was then replaced with fresh PMA-free media, and the cells
were left to rest for 24 h before the infection protocol. Cell differentiation
following PMA stimulation was monitored over time by microscopic observation
of cell morphology and plastic adherence properties.
gift from Prof. David Hume) were maintained in RPMI-1640 medium (Thermo
Fisher) supplemented with 10% heat inactivated fetal bovine serum
(FBS, Life Technologies) and GlutaMax (Thermo Fisher) at 37 °C
in a 5% CO2 environment. Preparation of THP-1 cells prior
to infection, THP-1 monocytes were differentiated into macrophages
by treatment with 50 nM phorbol 12-myristate 13-acetate (PMA, VWR)
for 3 days at a density of 5 × 104 cells per well
in a 96-well tissue culture plate (Thermo Fisher, Nunclon Delta).
The media was then replaced with fresh PMA-free media, and the cells
were left to rest for 24 h before the infection protocol. Cell differentiation
following PMA stimulation was monitored over time by microscopic observation
of cell morphology and plastic adherence properties.
3
Multicolor Flow Cytometry and Sorting
4
High-content imaging for drug screening
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
J-Lat 10.6 or 5A8 cells were seeded into 384-well black, glass-bottom plates (Greiner) using an Agilent BioTek Multiflo. All compounds were stored at 10 mM in DMSO, and 0.25 μl was added to each well using a Biomek Fx (Beckman Coulter). Media containing 0.25 μl DMSO or 5.0 ng/mL PMA was added to the first and last two columns, respectively. Cells were incubated for 24, 36, or 48 hours. Cells incubated with Hoechst (Invitrogen H3570) for 15 minutes then each plate was imaged using ImageXpress Micro Confocal High Content Imaging System (Molecular Devices). Images were analyzed using the MetaXpress Analysis software, and the raw data was imported into the Collaborative Drug Discovery vault online to generate Z scores. The EC50 was conducted similarly; each compound was purchased new, resuspended at 10 mM in DMSO, then a 10-step 4-fold dilution was pipetted down the plate. Follow up studies were done manually with IBET151 (Cayman Chemistry 11181), NSC95397 (Cayman Chemistry 21431), PMA (VWR 102515-692), prostratin (Cayman Chemistry 10272), SAHA (Cayman Chemistry 10009929), and Sunitinib (Cayman Chemistry 13159).
5
EMA/PMA Treatment for Selective DNA Extraction
6
Cytokine Profiling of PBMC Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We isolated PBMCs, challenged them ex vivo, and measured the cytokines and chemokines that were produced by the cells [46 (link), 47 (link)].
We conducted two studies: a “mycotoxin study” where the challenges were either a mycotoxin (SG), an aqueous S. chartarum extract with a mix of mycotoxins and other antigens (SST), a non-specific toxin (ionomycin [positive control, VWR, Radnor, PA]), or media alone (negative control), each with or without the adjuvant phorbol 12-myristate 13-acetate (PMA, VWR, Radnor, PA); and a “mold spore study” where the challenges were with intact mold spores, a non-specific toxin (phytohemagglutinin [PHA]), or media alone.
We conducted two studies: a “mycotoxin study” where the challenges were either a mycotoxin (SG), an aqueous S. chartarum extract with a mix of mycotoxins and other antigens (SST), a non-specific toxin (ionomycin [positive control, VWR, Radnor, PA]), or media alone (negative control), each with or without the adjuvant phorbol 12-myristate 13-acetate (PMA, VWR, Radnor, PA); and a “mold spore study” where the challenges were with intact mold spores, a non-specific toxin (phytohemagglutinin [PHA]), or media alone.
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!