All cell culture procedures were performed under sterile conditions in laminar class II biohazard safety cabinet (ESCO). The cell cultures were incubated at 37oC in 5% CO2 humidified incubators (RSBiotech).
MSC were cultured in MSC complete medium made up of Dulbecco’s Modified Eagle’s medium with nutrient mixture F-12 (HAM)[1:1] (DMEM/F12) with GLUTAMAX -I (Gibco, Invitrogen, USA), supplemented with 10% pre-selected foetal bovine serum (Stem Cell Technology Inc.), 1% of Penicillin /Streptomycin (Gibco, Invitrogen), 0.5% Fungizone (Gibco, Invitrogen), 0.1% Gentamicin (Gibco, Invitrogen), with or without 40ng/ml basic fibroblast growth factor (bFGF) (Peprotech, USA) . The serum used was pre-selected by the manufacturer to support MSC growth optimally in vitro culture while preserving MSC multi-potency to differentiate into chondro-, adipo- and osteogenic pathways.
3.3 T cell media
PBMC were cultured in complete T cell medium made up of RPMI 1640 (Gibco BRL, Invitrogen) supplemented with 10% foetal bovine serum (Gibco BRL, Invitrogen) or Human AB Serum and 1% Penicillin/Streptomycin (Gibco BRL, Invitrogen), 0.5% Fungizone (Gibco BRL, Invitrogen), 0.1% Gentamicin (Gibco BRL, Invitrogen).
3.4 Flowcytometry Analysis
3.4.1 Immunophenotyping
The surface markers of cells were determined by direct immunofluorescence staining and analysed by flowcytometer. All antibodies were listed in Table 1. Cells were harvested and washed with 0.5% of BSA in 1X PBS (phosphate-buffered saline). Aliquots of cells from 105 to 106 were labelled with anti-human monoclonal antibodies, for 20 minutes at 2-8oC and washed with 0.5% of BSA in 1X PBS. All antibodies are raised in mice against human epitopes. The fluorochrome analysis wa...
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...idine (3H-TdR) incorporation assays
Proliferation assays were measured by tritium thymidine (3H-TdR) incorporation which reveals the proportion of cells in S phase of the cell cycle. In brief, at final 18 hours of incubation, 3H-TdR (0.037 MBq/well [0.5 μCi/well]) (Pelkin Elmer) were added to pulse the cultures. At the end of incubation, the cells were harvested onto glass fibre filter mats A (Perkin Elmer) using a 96 well plate manual cell harvester (MACH IIIM-FM, Tomtec, Inc. Hamden, CT USA). Scintillation cocktail was added to amplify the signal. Liquid scintillation spectroscopy was counted by Microbeta Trilux beta counter (Pelkin Elmer).
3.6 Statistical Analysis
Values for all measurements are presented as mean ± SD or stated otherwise. Comparisons for all pairs were performed by Student’s t-test. Significance levels were set at p value of 0.05.
The thresholds used to calculate each mean were not highly variable between trials. The data recorded over each trial were highly consistent between one another, except for a slight deviation in the measurements recorded on the palm of the hand. During ascending trial three on the palm, the results deviated from the norm in reference two the two prior trails. On trial one and two, 0.05 was the only measurement that was not felt. On trial three, not only was 0.05 not felt, but 0.10 was also not felt, which deviated from the norm set forth in the two prior
The gene which is responsible for this disease, FGFR3, is located on chromosome 4 at 16.3, which is on the short arm near the telomere (4). Under normal circumstances, this gene forms fibroblast growth receptor 3 which interacts with a protein to begin a stream of signals that contribute to bone development and maintanence; it is also thought that this gene is also important in other tissue development (6, 7, 10-12). Some of the known pathways involved with FGFR3 are STAT1/3, STAT5, MEK1, ERK1, and MAP kinase signaling. Chondrogenesis and osteogenesis are two processes managed by these pathways and are greatly affected by a mutation (13-15). The sections of these pathways that involve and are affected by the mut...
Modern cytological work involves an intricacy of detail, the significance of which can be appreciated by the specialist alone; but Miss Stevens had a shre in a discovery of importance, and her name will be remembered for this, when the minutiae of detailed investigations that she carried out have become incorporated in the general body of the subject.
“Cells Involved In Immune Responses and Antigen Recognition.” Microbiology and Immunology. Web. 18 Dec. 2011. .
Currently, the limitations on research are too restricting, as researchers are limited to resources already gathered. There are sixty existing stem cell lines today, already derived from embryos. Researchers are to only use these lines. These limitations severely hinder stem cell research. The government, especially President Bush, should re-evaluate stem cell research.
..., M., Oort, F., & Sprangers, M. (2013). Significance, truth and proof of p values:
Bone tissue engineering (BTE) plays an important role in treating bone diseases related to osteoporosis and other orthopedic treatments. Although several methods are used in orthopedic surgery, some bone transport methods such as autografting and allografting have a certain number of disadvantages. Both are expensive methods and they can be exposed to infections and diseases. Therefore, in stead of using these potential risky methods, bone tissue engineering process are used to treat in orthopedic treatments. In general, both tissue engineering and bone tissue engineering have major constituents including stem cells, scaffold, bioreactors and growth factors.
Collected data were subjected to analysis of variance using the SAS (9.1, SAS institute, 2004) statistical software package. Statistical assessments of differences between mean values were performed by the LSD test at P = 0.05.
In the case of temperatures the cultures were incubated at each determined temperature. For the UV radiation, cells were exposed to UV light for 10 seconds and then grown in 30oC. For the EtBr treatment, 50ul of EtBr was added to the growth medium and cells were incubated at 30oC. In the case of sunlight exposure, cells were exposed to sunlight directly and grown at room temperature
2. The specific microorganism should be isolated from the diseased animal and grown in pure culture on artificial laboratory media.
Many factors can influence the results of testing this hypothesis. All variables have been controlled except for the variable gender. Both the male and female subjects are close in age (< two years difference), both are nonsmokers, both possess small body builds for their respective gender, and both have no debilitating medical conditions (e.g., asthma, diabetes, heart condition). Controlling these factors allowed for the testing of the hypothesis, which is focused strictly on gender.
This experiment consisted of 32 participants, of which 17 were male and 15 female, with a mean age of 19.8 (SD=0.87). Students selected from a variety of courses at the University of Aberdeen were recruited as participants.
In this method, living spores which are resistant to whichever sterilizing agent is being tested are prepared in either a self contained system, such as dry sp...
· Loughman, W.D., Sargent, T.W. & Israelstam, D.M. (1967, 27 October): Leukocytes of humans exposed to lysergic acid diethylamide: lack of chromosomal damage. Science. 158:508-510.
The field of regenerative medicine encompasses numerous strategies, including the use of materials and de novo generated cells, as well as various combinations thereof, to take the place of missing tissue, effectively replacing it both structurally and functionally, or to contribute to tissue healing[29]