Transfection cell density

Finally, estradiol induced a response in the luciferase activity of MVLN transfected cells. The hormone increased three-fold the basal level of enzymatic activity (Fig. 7.3.2). The luciferase activity of each experiment was normalised to the steroid-free control cultures to correct for differences in the initial seeding density. The activity induced by the antiestrogen RU 58 668 was lower than the response to the control. 

Cell microarrays have also been fabricated. Ziauddin and Sabatini (2001) demonstrated the ability to transfect cells cultured onto plasmid DNA arrayed in gelatin on a standard DNA microarray slide. Xu (2002) printed down cells in the form of high density microarrays on permeable membranes and demonstrated phenotypic assay performance with the immobilized cells. The commercialization of viable cell arrays will permit an even closer look at cell-mediated events during the drug discovery process. 

Wait at least 24 h after transfection to allow for sufficient expression of the resistance gene before adding antibiotics. Include two plates as controls one plate of transfected cells without antibiotic selection as positive control for cell viability and another plate of untransfected cells, seeded at the same density and treated with blasticidin to monitor cell resistance and antibiotic activity. All cells should die in this control under antibiotic selective pressure. 

The cells are examined on the day of transfection to make sure that they are at the optimal cell density (60-80%). Add the TransIT -LTl reagent (2-8 pL per 1 (jig DNA) directly into 250 jjlL of serum-free medium contained in a sterile polystyrene tube. Mix completely by gentle pipetting. Incubate the reagent at RT for 10 min and add plasmid DNA (pCI-luc or pLIVE-lacZ) (1-3 pg per well) to the diluted TransIT -LTl reagent and mix by gentle pipetting, followed by incubation at RT for 15-30 min (see Note 3). 

Approximately 24 h prior to transfection, plate cells at a cell density of 106 cells in complete growth medium per well of a 6-well plate and culture the cells overnight. 

Approximately 18-20 h prior to transfection, seed cells at a cell density of 5 x 105 cells per mL in serum-tree growth medium. Seed 25 x 106 cells in 50 mL of culture media in a 125 mL Coming Spinner Flask to reach approximately double the density overnight. 

Fig. 2. TransIT-LTl-mediated transfection of HeLa cells at varying cell densities HeLa cells were plated in 12-well plates and transfected in parallel at 50% and 90% confluency. TransIT-LTl reagent transfections were performed in duplicate using a luciferase expression vector (pCI-luc) and 3 iL reagent per well, shows the importance of plating cells at optimal density for transfection. Twenty-four hours post-transfection, cells were harvested and assayed for luciferase activity. Visual confluence (line graph) was measured under the microscope at harvest. The data represent the average luciferase activity (Relative Light Units - RLUs in Millions) from three experiments performed on different days.

Approximately 24 h prior to transfection, plate 6 x 104-2.4 x 105 cells per mL adherent cells in a T-75 flask, to attain an appropriate cell density of 60-80% confluence the following day. Incubate the cells overnight. 

Trypsinize the T-75 flask according to standard procedures (see Note 9) and add 14 mL of complete growth media to the T-75 flask. Mix cells thoroughly. Plate cells (500 xL per well of a 24-well plate) at an appropriate cell density (1.2 x 104—4.8 x 105 cells per mL) to obtain 60-80% confluence at transfection and incubate for 2-3 h to allow cells to adhere to the plate. 

Addition of transfection complex to cells below the recommended cell density could lead to cytotoxicity. Therefore, it is necessary to determine the optimal cell density for each cell type in order to reduce toxicity. 

Fig. 2. Representative HUVEC transfection efficacy of C32 and method for FACS gating. The one-dimensional histogram of C32/DNA transfected cells (56.5% positive) (left) includes some falsely positive cells that are excluded during the two-dimensional analysis of the same data set as shown by the FACS density plot gating for the negative control (0% positive) (middle) and the C32/DNA transfection (44.7% positive) (right). Ratio of GFP fluorescence (x-axis) to background fluorescence (y-axis) is used to accurately gate positive cells.

Make sure to have enough cells. Cell density (50-100) x 10 cells per ml is sufficient to perform FACS analysis. Perform FACS analysis as quickly as possible to avoid cell aggregation and aging in the FACS buffer. Cells are magnetically labeled post-magnetofection due to association with magnetic transfection complexes vortex cells before FACS analysis. 

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