AMac isolated from five pigs were tested for LDH release after stimulation with 1 μg each of Nagasaki liquid OMV, Nagasaki plate OMV, Nagasaki liquid sonicate, D74 liquid OMV and 10 μg E. coli 0111:B4 LPS (Invitrogen) LPS as a control. Purified OMV, whole cell sonicate or LPS were resuspended in room temperature supplemented media (RPMI 1640, 5% swine sera, 5mM HEPES, 1mM l-glutamine) at indicated concentrations. The supernatant was removed from AMac previously seeded into 48-well plates, and 500 μl of media with appropriate sample (OMV, sonicate, LPS) was added to the wells. Cells were incubated at 37°C, 5% CO2 for 18 h, supernatants were collected and clarified (200 × g, 5 minutes). A lactate dehydrogenase (LDH) assay (CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega) was performed according to manufacturer’s instructions. Background LDH release was measured in medium from cells stimulated with only media, while total LDH release (= 100% cytotoxicity) was measured from unstimulated cells that were lysed by freezing and thawing. The percentage of LDH release was calculated by subtracting the background LDH release value from the LDH release value of the treated cells, and this number was then divided by the total LDH release value and multiplied by 100.
E coli 0111 b4 lps
Manufactured by Thermo Fisher Scientific
Sourced in United States
E. coli 0111:B4 LPS is a laboratory reagent derived from the cell wall of Escherichia coli bacteria. It is commonly used in research applications to study inflammatory responses and immune system function.
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Lab products found in correlation
Cytotox 96 non radioactive cytotoxicity assay, by Promega (1 mentions)
Superscript vilo cdna kit, by Thermo Fisher Scientific (1 mentions)
Mag max express, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Elisa kit, by BD (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Colistin, by Merck Group (1 mentions)
4 protocols using e coli 0111 b4 lps
1
OMV-induced LDH Release in Porcine Alveolar Macrophages
2
Transcriptional Profiling of Immune Cells
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AMac were stimulated with purified OMV (1 μg), whole cell sonicate (1 μg), live H. parasuis (MOI 2:1) or E. coli 0111:B4 LPS (10 μg/ml) (Invitrogen). After 18 h incubation, media was collected and cells were lysed on the culture plate with TRI reagent (Ambion). RNA was isolated using a MagMax Express (Life Technologies) and cDNA was synthesized using a SuperScript VILO cDNA kit (Invitrogen). SYBR Green-based real-time PCR was conducted for various mRNA targets using SYBR Green Master mix (Life Technologies) following manufacturers’ protocol. All samples were run in duplicate. Levels of mRNA were calculated using the threshold cycle 2-ΔΔCt method, which expresses mRNA in treated cells relative to non-stimulated cells after normalizing to β-actin [54 (link)]. Primers used were previously reported [55 (link)] and PCR products were < 100 bp in size.
3
LPS-Induced Cytokine Release Assay
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This experiment
was performed following a previously published protocol.51 (link) Briefly, hPBMCs were freshly isolated and seeded
in 24-well plates for 3 h [1 × 106 cells in 1 mL of
Roswell Park Memorial Institute medium (Gibco)]. To these wells, 20
ng/mL of E. coli 0111:B4 LPS (Molecular
Probes, Life Technologies, USA) was added in the presence or absence
of NCK-10 (10 μg/mL). Hanks’ balanced salt solution and
only NCK-10 were also added as controls. After incubation for 18–24
h, ELISA was performed on the cell culture supernatants for cytokines
TNF-α and IL-6. ELISA kit was bought from BD Biosciences, and
manufacturer’s instructions were followed for the experiment.
was performed following a previously published protocol.51 (link) Briefly, hPBMCs were freshly isolated and seeded
in 24-well plates for 3 h [1 × 106 cells in 1 mL of
Roswell Park Memorial Institute medium (Gibco)]. To these wells, 20
ng/mL of E. coli 0111:B4 LPS (Molecular
Probes, Life Technologies, USA) was added in the presence or absence
of NCK-10 (10 μg/mL). Hanks’ balanced salt solution and
only NCK-10 were also added as controls. After incubation for 18–24
h, ELISA was performed on the cell culture supernatants for cytokines
TNF-α and IL-6. ELISA kit was bought from BD Biosciences, and
manufacturer’s instructions were followed for the experiment.
4
Synthesis and Characterization of Colistin Liposomes
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The synthetic protocols used
for the preparation of the compounds have been described in previous
publications.38 (link) Colistin was bought from
Sigma-Aldrich (Bangalore, India). All lipids were purchased from Avanti
Polar Lipids (Alabaster, AL, USA). Escherichia coli BODIPY-LPS [BODIPYFL (503/513)] and E. coli 0111:B4 LPS were obtained from Molecular Probes, Life Technologies,
USA. P. aeruginosa strain PA R590 was
acquired from the Department of Neuromicrobiology, National Institute
of Mental Health and Neuro Sciences, Hosur Road, Bangalore 560029,
India. Culture media were from HiMedia and Sigma-Aldrich (India).
SYTO-9 was bought from Thermo Fischer, Bangalore, India.
for the preparation of the compounds have been described in previous
publications.38 (link) Colistin was bought from
Sigma-Aldrich (Bangalore, India). All lipids were purchased from Avanti
Polar Lipids (Alabaster, AL, USA). Escherichia coli BODIPY-LPS [BODIPYFL (503/513)] and E. coli 0111:B4 LPS were obtained from Molecular Probes, Life Technologies,
USA. P. aeruginosa strain PA R590 was
acquired from the Department of Neuromicrobiology, National Institute
of Mental Health and Neuro Sciences, Hosur Road, Bangalore 560029,
India. Culture media were from HiMedia and Sigma-Aldrich (India).
SYTO-9 was bought from Thermo Fischer, Bangalore, India.
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