Project description:We examined the effects of ligating CD4 expressed by primary human peripheral blood monocytes with soluble MHC Class II. 3 replicates are included. Fresh blood monocytes, obtained through negative selection via magnetic activated cell sorting (Miltenyi, Auburn, CA), from three separate individual donors were treated with RPMI-1640 medium alone or with medium containing sMHC II (1 ug/ml) for three hours. Cells were harvested and RNA purified using the RNeasy procedure (Qiagen). RNA was then analyzed by the UCLA Clinical Microarray Core Laboratory using the Human U133 Plus 2.0 Array (Affimetrix). Data was analyzed using Dchip software. Visual inspection did not find any potential outliers or suspect areas. Data across all groups were normalized using the PM/MM difference.
Project description:Cultured monocytes were divided into three groups; 1) a control group 2) monocytes treated with LPS (0.5 ug/ml) for 6 hours 3) monocytes pre-treated with 100nM 9CisRA for 1 hour followed by LPS treatment for 6 hours.
Project description:The goal of this experiment is to profile LPS-stimulated transcriptome changes in human macrophages. Human whole blood was from the Sanofi in-house blood donor service that is approved by the local ethics committee and all blood donors signed informed consent. Human peripheral blood monocytes were isolated from anonymous donors’ prefinal blood using Ficoll density centrifugation, followed by magnetic separation with positive selection (CD14 MicroBeads, Miltenyi Biotec). Monocytes were differentiated into macrophages by culturing in macrophage serum-free medium (Life Technologies) containing 50 ng/ml recombinant human macrophage colony-stimulating factor (Immunotools) for 5 days. Following differentiation, cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (Thermo Fisher) and maintained at 37°C in a 5% CO2/air environment. Macrophages were stimulated with 50 ng/ml lipopolysaccharides from Escherichia coli O111:B4 (Sigma) for 24 hours.
Project description:RNA-seq libraries were constructed for three samples, including (I) ΔURA3 strain grown in SD medium containing 2% w/v glucose(pH=5.0);(II)ΔURA3 strain grown in SD medium containing 2%w/v glucose and 2% w/v succinic acid(pH=5.0);(III)ΔURA3 strain grown in SD medium containing 2% w/v succinic acid(pH=5.0). For preparation of RNA samples, ΔURA3 cells grown overnight were inoculated into 50 ml liquid Synthetic Dextrose (SD) medium with the initial inoculation amount of OD600= 0.1, and cultured for 24 hours to the logarithmic growth phase (OD600= 2-3). The cells were collected by centrifugation at 6000g for 5 minutes. After washing twice with phosphate buffer saline (PBS), they were inoculated into new 50 mL SD medium containing carbon sources of different combinations of glucose and succinic acid .The pH of the culture sample is adjusted to 5.0 with NaOH. After flask culturing at 30°C and 250 rpm for an additional three hours, the yeast cells were collected by centrifugation for total RNA isolation and Illumina RNA-seq library construction. Total RNA for samples were isolated using TRIzol reagent (Invitrogen, Grand Island, USA), then used for high-throughput RNA sequencing. The 150-nt paired-end RNA-seq libraries were generated commercially at Novogene Biotechnology Co. Ltd (Tianjin, China) by using Illumina’s Hiseq X Ten platform (Illumina, San Diego, USA). Each sample contains two biological replicates.
Project description:To investigate the possible therapeutic effects of ibuprofen and Pep19-2.5 alone or in combination on the LPS-response of human monocytes. Human monocytes were challenged with 0.1ng/ml LPS and/or 10ng/ml Pep19-2.5 LPS. Medium controls served as unstimulated samples. LPS and Pep19-2.5 was used either alone or together to LPS-challenged samples. RNA was extracted after 4 hrs of stimulation. Three biological replicates were conducted
Project description:To explore the full extent of IFN-regulated transcriptional changes, we exposed monocytes from two healthy donors to recombinant type I IFN (IFN-M-NM-12b) in vitro. RNA was extracted at different incubation times (1, 6, 24, 48 and 72 hrs) and the expression data was normalized to that of monocytes cultured with medium. Blood monocytes isolated from healthy volunteers were incubated with 20% autologous serum alone or in the presence of 1000 U/ml of IFNM-NM-12b (Schering Plough, Kenilworth, NJ) in 6-well plates at a concentration of 106 monocytes per well in 3 ml of media. After incubation for one hour at 37 M-bM-^AM-0C, cells were harvested and RNA was extracted. Identical experiments were done after the following incubation time points: six hours, twenty four hours, two days, and three days.
Project description:Human T-cells were isolated from full blood by ficoll centrifugation and subsequent negative depletion with magnetic beads of monocytes and B-cells and treated with 5ug/ml PHA for 24 hours.
Project description:In this study, the effect of nitroprusside on protein expression of the model sulfate reducing bacterial isolate (Desulfovibrio vulgaris Hildenborough) was investigated. Three different experiments were done with different exposure time to nitroprusside (30 min, 1 hour, and 3 hours). In each experiment, the culture was grown in quadruplet of serum bottles containing 40 ml complex medium (Postgate medium B) under anaerobic condition at 30oC. At mid-log growth phase, each of the quadruplet culture was divided into two (20 ml each) and nitroprusside (0.25 mM) was added to one of each bottle while the other bottles were used as controls. After 30 min, 1 hour, and 3 hours of exposure to nitroprusside, the culture (1 ml) was pelleted and used for proteomic analysis.
Project description:Peripheral blood samples from children enrolled in the URECA cohort were collected at age 2. In short, at each URECA research center, within 16 hours of blood collection, mononuclear cells were separated and incubated (48 hours, 37C, 5% CO2) in the presence of German cockroach extract (10mg/mL; CR), dust mite extract (Dermatophagoides pteronyssinus, 10mg/mL; DM), tetanus toxoid (5mg/mL; TT), or medium alone (NS).
Project description:To explore the full extent of IFN-regulated transcriptional changes, we exposed monocytes from two healthy donors to recombinant type I IFN (IFN-M-NM-12b) in vitro. RNA was extracted at 6 hrs and the expression data was normalized to that of monocytes cultured with medium. Blood monocytes isolated from healthy volunteers were incubated with 20% autologous serum alone or in the presence of 1000 U/ml of IFNM-NM-12b (Schering Plough, Kenilworth, NJ) in 6-well plates at a concentration of 106 monocytes per well in 3 ml of media. After incubation for six hours at 37 M-bM-^AM-0C, cells were harvested and RNA was extracted.