Culture plates and flask

Fibroblast cultures for each patient and control are grown to confluency in T-75 culture flasks. The assay is then performed in triplicate using six-well plates. First, 1 ml of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Sigma T4049) is added to each T-75 flask. After the cells have lifted from the flask, they are resuspended in 6 ml complete MEM. A 1-ml aliquot of the cell suspension is transferred to a 50-ml conical tube containing 14 ml of MEM complete medium (see above). Following further resuspension, 4 mL is plated into each of three wells in a six-well culture plate and placed into an incubator. Once the cells have attached to the seeded plates and are approximately 90-100% confluent (3-4 days), complete MEM is replaced with 1.5 ml of complete IVPM (see above). 

Gelatin-coated culture plates or flasks Coat culture plates or flasks for 2 h with 0 1 % gelatin/PBS solution Aspirate off and wash once with PBS. Cells can be plated imraediumtely or, alternatively, the plates or flasks can be stored in PBS until needed... 

Althou hybridoma cells are spherical and well adapted to suspension culture they do grow well in stationary culture resting on the substrate, sometimes with very light attachment. This means that a wide selection of culture vessels and systems are available for their culture ranging from a simple tissue culture plate or flask to highly sophisticated bioreactors with full instrumentation to control the physiological environment (1-3). Commercially hybridoma cell lines are grown at scales up to 2000 litres and beyond in culture units scaled-up from laboratory size vessels. Laboratory, pilot, and production scale is ill-defined so in this chapter laboratoiy scale will be taken as 10 litre volume cultures, and below. 

All necessary materials should then be placed in the cabinets, segregating clean items, cultures, and receptacles for contaminated items. The worker should organize the materials so that contaminated materials do not need to pass over clean items (remember, the air flow direction is downward). A good layout of materials would place cultures, clean pipets, flasks, etc. toward the front on either side of the work area, and a discard tray for culture plates and pipets towards the rear of the work space, as shown in Figure 9.9. Avoid placing items within ten centimeters of the cabinet front. The cabinet must not be overloaded with materials. Overloading will disrupt the air stream and interfere with proper cabinet performance. 

Culture medium osmolality can also increase due to evaporation, since culture flasks are generally not sealed so as to allow equilibrium between culture medium and the C02-air gas mixture. A slightly hypotonic culture medium can be more adequate for open cultures in multiple well plates or in Petri dishes to compensate for evaporation during incubation. To avoid large variations in osmolality during culture, the relative humidity of the culture environment should be maintained near to saturation. 

Fig. 3.2(a). Vessels in which cells may be cultured. At the top left are three sizes of flask (125, 75 and 25 cm2) and at the bottom left three sizes of dish (9.5 and 3 cm diameter). To the right are shown 6, 12 and 24-well tissue culture trays and a 96-well microtitre plate. At the bottom right is an 8-chamber culture slide, (b) Millicell inserts for 6-well TC trays provide a semi-permeable growth surface. 

The growth vessel and some supplements are determined by whether the cells are grown attached or in suspension. When the cells are anchorage dependent, they can be grown in plates or flasks. As serum is reduced, it may be necessary to add attachment factors to the culture. For cells grown in suspension in spinner flasks or fermenters, non-ionic surfactants (F68) that increase viscosity may be needed to minimize shear stress caused by agitation. 

Because most routine cell types were originally grown on glass, the first commercially available tissue culture surface was modelled after glass chemistry. Conventional tissue culture surfaces therefore are hydrophilic and have an oxygenated chemistry and a net-negative surface charge. This chemistry is basically the same whether the treatment process is produced by corona or plasma. This is the routine surface that is commercially available from a number of different suppliers on plastic dishes, flasks, plates and roller bottles. 

Coating of Cell Culture Plates, Flasks, and Filters... 

Shut off the hot plate and the air supply. Rinse all of the pipets with acetone and store them in the 125 mL Erlenmeyer flask. Empty the culture tubes into a waste container, rinse them with acetone, and store them for future use. You may take the column home as a token of our friendship. 

Note the monocytes will differentiate into adherent macrophages by day 7 of incubation. To harvest cells, discard the culture medium and wash once with 25 mL of cold PBS without Ca++ or Mg++. Add 20 mL of fresh cold PBS to the flask and incubate on ice until cells begin to round-up and detach (usually 15-20 min) (see Note 14). Gently scrape cells into the PBS with a cell scraper and transfer the cell suspension to a 50-mL conical centrifuge tube. Pellet the cells by centrifugation at 500 x g for 5 min. Resuspend cells in 5-10 mL of RPMI plus 10% FBS and 50 ng/mL M-CSF. Count cells, add medium to adjust to the desired cell concentration, and re-plate in a tissue culture plate (see Note 15). Incubate at 37 °C in 5% CO2 for at least 6 h to allow macrophages to adhere. 

Tissue culture-treated plastic flasks, dishes, and 24-well plates. 

The minimum volume of cell culture which would provide sufficient aeration for good cell growth while still maintaining a reasonable extraction volume was determined over a range from 50 mL in the Erlenmeyer flasks down to one mL in a 96-well plate. The final volume which gave the correct balance between having sufficient aeration and amount of cell culture for metabolism was 7 mL of cell culture in a 6-well culture plate. 

Uptake and fixation of Cj - in the cyanobacterium were determined by silicone oil centritu tion method (4). Transformation of the mutant C3P-0 was performed as in (5). The cells were grown in a flat flask (50 ml) with aeration of 5% CO2 at 30 C and harvested with the turbidity at 720 nm of 0.1 to 0.2. They were washed with and resuspended in the culture medium. The suspension was divided into 400 pi, to which 1 to 10 pg of the donor DNA was added. The mixture was shaken in the dark for 20 hours at 30 C. Portions (150 pi each) of the suspension were spread on agar plates and incubated at 42 C in ordinary air with continuous illumination. Discrete colonies became visible in 10 days, of incubation. 

Tissue culture flasks (T-75, Cat. No. 10-126-41), six-well tissue culture plates (Cat. No. 08-772-lB), sterile polypropylene conical tubes (50-ml capacity, Cat. No. 05-538-55A), sterile polypropylene round-bottom tubes (5-ml capacity. Cat. No. 14-959-lOA), and sterile conical-bottom microfuge tubes (1.5-ml capacity. Cat. No. 05-664-63) are obtained from Fisher Scientific. Disposable 1-ml syringes (Cat. No. 309602) and 30-gauge hypodermic needles (Cat. No. 5106) are purchased from Becton-Dickinson. A Hamilton microliter syringe (50-//1 capacity. Cat. No. 80501) is obtained from Hamilton Company. Micropipette tips (MC-50) are purchased from West Coast Scientific. The hand-held pipettor (Cat. No. P20) is obtained from Gilson Medical Electronics. 

Liquid overnight culture add ampicillin and chloramphenicol to a 250-mL Erlenmeyer flask containing the 50 mL of LB medium Remove a single bacterial colony from one agar plate and add it to the flask. Shake at 37°C overnight. 

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