A total of 2 × 105 cells/well (BMDM or J774) were seeded into a 96-well plate for Salmonella intracellular growth assays and incubated two hours to allow adherence to the plate. In some experiments recombinant murine interlukin-10 (Peprotech) (rIL-10) was added either two hours before or at the time of infection. Cells were inoculated at a range (100-3.125) of multiplicities of infection (MOI) the ratio of bacteria to eucaryotic cells. Infection was facilitated by centrifugation at 300 × g for 5 min. Cells were incubated for one hour with bacteria, and the medium was then removed. Fresh medium containing 50 μg/mL gentamicin (Sigma) was added to kill extracellular bacteria. One hour after gentamicin addition, medium was removed, and the cells were washed twice before the addition of fresh antibiotic free medium. All culture medium used in the arabinose inducible promoter system experiments contain 0.2% arabinose as indicated in the figures. To determine intracellular growth, medium was removed at indicated time points post-infection, and 1 mL of PBS was added to the cells. Cells were removed from the plate by vigorous pipetting. Cells were lysed by vortexing at maximal speed for one minute. Serial 1:10 dilutions of the lysate were made and plated onto LB agar. The resulting colonies were counted 24 hours later.
Ril 10
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RIL-10 is a laboratory equipment designed for research purposes. It functions as a refrigerated incubator, providing temperature and humidity control within a regulated environment.
Automatically generated - may contain errors
Lab products found in correlation
Gentamicin, by Merck Group (1 mentions)
Ril 13, by Thermo Fisher Scientific (1 mentions)
Lineage cell depletion kit, by Miltenyi Biotec (1 mentions)
Fitc conjugated anti cd34 mab, by BD (1 mentions)
Pe conjugated anti cd31 mab, by BD (1 mentions)
Kmg 2, by Lonza (1 mentions)
Cryptotanshinone, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
3 protocols using ril 10
1
Intracellular Growth Assay for Salmonella
2
Isolation and Culture of Human Epidermal Keratinocytes
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Human β-defensin-1 (HBD-1) kits were purchased from PeproTech (Rocky Hill, NJ, USA). HBD-1, murine β-defensin-1 (MBD-1), anti-HBD-1, and anti-MBD-1 antibodies were purchased from Alpha Diagnostic International (San Antonio, TX, USA). Lineage cell depletion kits were purchased from Miltenyi Biotec (Auburn, CA, USA). FITC-conjugated anti-CD34 mAb and PE-conjugated anti-CD31 mAb were purchased from BD Biosciences (Franklin Lakes, NJ, USA) and BioLegend (San Diego, CA, USA), respectively. Recombinant (r) CCL2, rIL-4, rIL-10, and rIL-13 were obtained from PeproTech. mAbs directed against CCL2, IL-10, and IL-13 were purchased from BioLegend. Adult normal human epidermal keratinocytes (NHEK) were obtained from Lonza (Walkersville, MD, USA) and propagated in a serum-free keratinocyte growth medium (KMG-2, Lonza) at 37°C. NHEK that underwent a second passage with KMG-2 were stored in liquid nitrogen. NHEK grown from the stored cells (the third passage) were used in this study. RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) was used as culture media for lineage−CD34+ cells.
3
Endometrial Cancer Cell Response to IL-10
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Ishikawa (a well-differentiated human endometrial adenocarcinoma cell line) and BeWo (human choriocarcinoma cell line) cells were maintained in DMEM/F12 and RPMI culture media, respectively, supplemented with 10% fetal bovine serum (Gibco, BRL/Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (HyClone, South Logan, UT, USA). After serum starvation for 12 h, Ishikawa cells were treated with different concentrations (0-100 ng/ml) of rIL-10 (Peprotech, Burlington, NC, USA) for different times (0.5-48 h). To inhibit the STAT3-dependent signaling pathway, Ishikawa cells were treated with 4.6 μM cryptotanshinone (Sigma, St. Louis, MO, USA) for 24 h before rIL-10 treatment.
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