Week #8: Scaling up—More the Merrier!
Turhan K -
Welcome back everyone!
This week marked a significant transition in my research journey: scaling up the HEK293T cultures from 10cm plates to the larger 15cm plates!
Since the initial stages of the project, I used 10cm plates, in which the number represents the diameter, to cultivate cells within. Without consuming a relatively large amount of materials (e.g. DMEM, a liquid used as a cell media/food for HEK293T cells), this size allowed for extended growth of the cells—often reaching 1 x 10^6 or higher cells at 80%-90% confluency— and a sufficient quantity of cells for my previous calcium-phosphate transfections.
However, after the results extracted from the previous transfection, the direction of my project shifted. At last, determining the optimal NS1-to-reporter plasmid ratio (1:1) based on last week’s experiments and previous, consistent results answered the preliminary question of my research: what conditions are appropriate to create a valid experiment that can express a sufficient sample of NS1 bound to human protein, in order to analyze interactions? Yet, with this question now satisfied, I’m moving toward the protein interaction studies, the heart of my project. I’ll need substantially more HEK293T cells and expressed NS1 protein than my previous 6-well plates or 10cm dishes could provide, to capture the interactions on the microscopic level. The 15cm plates offer approximately 2.25 times more surface area than the 10cm plates, which means more cells, more protein expression, and ultimately more of a sample to analyze underneath a Western Blot and mass spectrometry.
The initial transition to 15cm plates has proven successful! I adjusted certain aspects of cell maintenance, proportionate to the 3:2 ratio between the new and old plates. For instance, in lieu of the 10 mL of DMEM utilized to maintain 10 cm plates, I’m now pipetting approximately 17-25 mL of cell media into the 15 cm plate. However, other elements of growing HEK293T cells remained the same, such as the average wait time before splitting cells (3-4 days).
I’m looking forward to sharing the results of these larger-scale experiments in next week’s post. Thank you for reading!
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