Ex cell acf cho medium

On the day of the experiment, cells plated at a density of 2.9 × 10⁴ cells/cm² 72 h prior should be ∼70%–80% confluent. After cells were rinsed with prewarmed 1 × PBS (Ca²⁺/Mg²⁺ free), 3 mL of room temperature Detachin solution (Genlantis) was added to the flask and tilted gently two to three times to cover all the cells. Approximately 2 mL of Detachin was aspirated from the flask, leaving 1 mL on the cells, and then placed in the 37°C incubator for 5 min. Once cells had rounded up, the cells were dislodged by tapping the flask gently. The cells were then resuspended in 5 mL of warm serum-free media (EX-CELL ACF CHO Medium; Sigma; supplemented with 4 mM l-glutamine and 10 mM HEPES). A glass Pasteur pipette with a rubber bulb was then used to gently dissociate the cells from one another. Detachin will often leave cells as doublets or clumps, conditions that will disrupt seal formation on the QPatch. The cells were triturated ∼30 times and then, still using the Pasteur pipette, transferred into the QPatch Cell Hotel. This ensures that cells detach from one another leaving a suspension of single cells. A QStirrer was added to the Cell Hotel and the cell suspension was immediately placed on the QPatch device. Cells were utilized within 15 min.

Hela S3 cells were cultured in DMEM, supplemented with 10% FCS and 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C with 95% humidity and 5% CO₂. Human embryonic kidney EcoPack 2-293 cells (Clontech) were cultivated on collagen-coated (Collagen R; Serva Electrophoresis) plates under the same conditions. HEK293 cells were cultured under the same conditions. For protein purification, HEK293 cells were grown in EX-CELL ACF CHO Medium (Sigma-Aldrich, C5467) supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C with 95% humidity and 5% CO₂. CHO K1 cells were cultured in α-MEM medium supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 95% humidity and 5% CO₂. All cell lines used in this study were received from the Leibniz Institute DSMZ (German collection of microorganisms and cell cultures GmbH). For human cell lines, their identities were confirmed by STR profiling. For CHO cells, identity and purity were analyzed by a multiplex cell contamination test (Schmitt and Pawlita, 2009 (link)). All cell lines tested negative for mycoplasma contaminations.