FITC Labeling Of Purified Using Immunoaffinity Chromatography : Case Study

The large scale production of therapeutic proteins in industries is done in suspension culture as the adherent cell line poses a challenge in scaling up. Currently there are two dominant modes of operation for producing therapeutic proteins based on mammalian cell lines (Hu and Aunins, 1997) in industrial scale:

Tissue sample presented with large population of B cells, labelled with CD20, in follicles and macrophages labelled with CD68 were also visible in the intrafollicular zone, both are indicative of a humoral immune response.

15 cell lines were acquired for the purpose of this experiment. Process of subcellular compartment fractionation was performed on these cell lines, this included complete cells, nucleus and cytosol (seen in

Aspirate the DMSO medium, and re-suspend the cells belts in 20 ml media & incubate overnight.

A cell suspension containing 0.5 -1.0 x 10^6 cells/ml in media 10% FBS (Fetal Bovine Serum) was placed on a 24- well plate so a monolayer can be created at the bottom of the wells. The first step will consist of the lab student aspirating the media from the wells making sure to disrupt the monolayer of cells at the bottom of the wells. Next the wells will be washed with PBS to remove dead cells and debris. After the wells are washed 1ml of media (DMEM) in a 5% concentration will be added to keep the cells from dehydrating. 5% Media is used because the goal here is to allow migration and minimize proliferation. Next the lab student will create a horizontal “scratch” along the bottom of the well using a P200 pipette. The lab student should use enough force to crate the scratch but not too much because too much force will create uneven scratches. Finally the treatments will be added to the wells. The plate will then be placed under the light microscope and pictures of the scratches will be taken which will be labeled as (T0) Time point zero. Other pictures will be taken 24 hrs. later at (T24) time point 24. The photos will then be analyzed and compared the lab group to determine if the is any cell

DCs pulsed with inactive yeast cells were not able to induce maturation of DC in vitro and hence these cells were unable to develop Th1 response in vivo. Because of this, memory cells responsible for anticandidal protective immunity cannot be developed.

Preparation of microELISA plates for the detection of antibodies in sera from pre-immunized mouse against rabbit protein and comparing this to sera extracted at weeks 1 and 2 after immunization and re-immunization of the mouse to check for increased titer of antibodies in the sera

When temperature is lowered to 42°C, primers anneal to complimentary target RNA. After multiple rounds, amplification of cDNA can be quantified and analyzed. The amount of amplified DNA can also be seen on an agarose gel with an ethidium bromide stain.

Following extracts treatment, cells were trypsinized and washed twice in phosphate-buffered saline (PBS); cell cycle phases were then analyzed as described (Nicoletti et al., 1991) with a minor modification. Briefly, cells were fixed at 4°C overnight in 70% ethanol. After washing with Ca2+-Mg2+-free Dulbecco's PBS, cells were treated with 0.1 µg/ml RNase (Type I-A, Sigma), stained with 100 µg/ml propidium iodide (PI; Sigma) for 20 min, filtered, and kept on ice until measurement. Cells were measured by the FACSCaliburTM (Becton Dickinson) and the data obtained were then analyzed using ModFitsoftware. Cell fractions with a DNA content lower than

When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialized function. Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialized cells that can renew themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special

Stem cells have the remarkable potential to develop into any functional cell in the body during its early life and growth, additionally in the many tissues of the human body, they serve as a form of internal support and repair system dividing without limit as long as the person or animal is alive. Stem cells are distinguished from other cell types by two crucial characteristics, first, they are unspecialized cells capable enough of renewing itself through cell devision, sometimes after long periods of inactivity and secondly, under certain experimental or physical conditions, they can be induced to become a form of tissue or organ specific cells with certain unique functions. (stem cell basics, corporate author,

MSCs are a promising tool for cell therapy. They partly contribute to the repair of injured tissues, but they cannot effectively participate in tissue repairing because of their scarce number (e.g. 1 per 106 cells in bone marrow or 1 per 104 cells in umbilical cord) (6). Therefore, in vitro cultivation and expansion and increasing their number for therapeutic purposes is necessary. However, their characteristics and phenotypes may be affected after cultivation and proliferation, depending on factors such as age and donor conditions, cell separation techniques, culture media and supplements (8). Because of self renewality and the ability to differentiate into different lineages they are an extremely

RNA extraction Total RNAs were extracted from B. bovis-, B. bigemina-, and B. microti-infected RBCs using a commercial RNeasy Mini Kit (Qiagen, Germany). RNA samples were treated with RNase-free DNase I at 37 C for 1 h and purified again using the RNeasy Mini Kit (Qiagen, Germany). The concentration of the purified total RNAwas quantified using a Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., USA). The cDNAwas prepared from about 5 mg of the total RNA using the SuperScript® III First-Strand Synthesis System (Thermo Fisher Scientific, Inc., USA). 2.3.

MSCs are a promising tool for cell therapy. They partly contribute to the repair of injured tissues, but they cannot effectively play role in tissue repairing because of their scarce number (e.g. 1 per 106 cells in bone marrow or 1 per 104 cells in umbilical cord) (6). Therefore, in vitro cultivation and expansion and increasing their number for therapeutic purposes is necessary. However, their characteristics and phenotypes may be affected after cultivation and proliferation, depending on factors such as age and donor conditions, cell separation techniques, culture media and supplements (8). Because of self renewality and the ability to differentiate into different lineages they are an extremely appropriate treatment option (37) and are considered as an ideal source for tissue engineering (38).

The BM-MSCs were cultured in T25 flask culture (Orange Scientific, Braine-l 'Alleud, Belgium) with DMEM medium and incubated for reaching to 60-70% confluency. In the next step, DMEM medium was replaced with RPMI-1640 medium without FBS and was incubated for 72 hours (hrs). After incubation; medium was collected, cells and debris were removed by centrifugation. Supernatant was concentrated by 10 kDa MW cut-off ultrafiltration