Aliquots of thawed PBMCs from the EpiTOF experiment described above were washed and resuspended in RPMI 1640 (Corning, 10–040-CV) containing 10% FBS (Corning, 35–011-CV) and 1x Antibiotics/Antimycotics (Lonza, 17–602E) [complete media abx] at 4×10^6 cells/mL. 100 μL of cell solution were added to each well of a 96-well round-bottomed tissue culture plate and mixed with 100 μL of either complete media abx (unstim), a cocktail of synthetic TLR ligands mimicking bacterial pathogens (bac: 0.025 μg/mL LPS, 0.3 μg/mL Flagellin, 10 μg/mL Pam3CSK4), or a cocktail of synthetic TLR ligands mimicking viral pathogens (vir: 4 μg/mL R848, 25 μg/mL pI:C). Depending on cell numbers, PBMCs from each sample were stimulated with all 3 conditions in duplicate. After 24h of incubation at 37C and 5% CO2, cells were spun down, supernatant was carefully transferred into new plates, and immediately frozen at −80C until further analysis using Luminex.
Antibiotics antimycotics
Manufactured by Lonza
Lonza's Antibiotics/Antimycotics product is a sterile solution designed for use in cell culture applications. It is a mixture of antibiotics and antimycotics that helps prevent bacterial and fungal contamination in cell cultures. The product is available in various concentrations and package sizes to meet the needs of different research and manufacturing applications.
Automatically generated - may contain errors
2 protocols using antibiotics antimycotics
1
Stimulation and Analysis of PBMCs
2
Isolation of Mononuclear Cells from Ileum and MLNs
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To isolate mononuclear cells (MNCs) from the ileum and MLNs, tissues were minced and digested in RPMI-1640 medium (Lonza) supplemented with 2 mg/mL collagenase/dispase (Roche Diagnostics) and 5% fetal bovine serum (FBS) (Lonza) at 37 °C with periodic agitation for 40 minutes. The cell suspension was sequentially passed through 100-μm and 40-μm cell strainers (BD Biosciences) and centrifuged at 500 X g for 10 minutes. The cell pellet was resuspended in RPMI-1640 medium (Lonza) containing 5% FBS (Lonza) and layered on an OptiPrep (Stemcell Technologies) density gradient. Following centrifugation at 750 X g for 30 minutes with the brake disengaged, the light density fraction was collected and washed twice with ice-cold PBS. The washed cells were resuspended in RPMI-1640 medium (Lonza) supplemented with 5% FBS (Lonza) and 2% antibiotics-antimycotics (Lonza). Cell viability and counts were determined by trypan blue dye exclusion using a hemocytometer.
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