CpGrich

Protocol tips

Upstream tips	Protocol tips	Downstream tips
CHO K1 cells (American Type Culture Collection, Manassas, VA) were cultured in tissue culture plates using Dulbecco’s modified Eagle’s medium (DMEM) + GlutaMaxTM (Life Technologies) supplemented with 10 % fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO). Cells were passaged every 3 to 4 days by diluting cells to 2 × 105 cells/mL. Cell density and viability were measured using the trypan blue exclusion method on a Vi-cell XR cell viability analyzer (Beckman Coulter, CA).	Three separate transfections were performed using each promoter using the Nucleofector I system from Lonza (Cologne, Germany). 5 × 106 cells were transfected with 5 μg of linearized plasmids in each transfection. The transfected cells were transferred to 6-well tissue culture plates containing DMEM supplemented with 800 μg/mL of G418 (Sigma-Aldrich) for selection 24 h after transfection.	Fluorescence-activated cell sorting (FACS) analysis was also performed using FACS Calibur (Becton Dickinson, Franklin Lakes, NJ) to determine the transient expression obtained using each promoter. Upon recovery of the stably transfected pools, at least nine clones were randomly selected from each stable pool by limiting dilution for a total of 30 clones to be carried forward for stability tracking.

Upstream tips
CHO K1 cells (American Type Culture Collection, Manassas, VA) were cultured in tissue culture plates using Dulbecco’s modified Eagle’s medium (DMEM) + GlutaMaxTM (Life Technologies) supplemented with 10 % fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO). Cells were passaged every 3 to 4 days by diluting cells to 2 × 105 cells/mL. Cell density and viability were measured using the trypan blue exclusion method on a Vi-cell XR cell viability analyzer (Beckman Coulter, CA).
Protocol tips
Three separate transfections were performed using each promoter using the Nucleofector I system from Lonza (Cologne, Germany). 5 × 106 cells were transfected with 5 μg of linearized plasmids in each transfection. The transfected cells were transferred to 6-well tissue culture plates containing DMEM supplemented with 800 μg/mL of G418 (Sigma-Aldrich) for selection 24 h after transfection.
Downstream tips
Fluorescence-activated cell sorting (FACS) analysis was also performed using FACS Calibur (Becton Dickinson, Franklin Lakes, NJ) to determine the transient expression obtained using each promoter. Upon recovery of the stably transfected pools, at least nine clones were randomly selected from each stable pool by limiting dilution for a total of 30 clones to be carried forward for stability tracking.

Publication protocol

"CHO K1 cells (American Type Culture Collection, Manassas, VA) were cultured in tissue culture plates using Dulbecco’s modified Eagle’s medium (DMEM) + GlutaMaxTM (Life Technologies) supplemented with 10 % fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO). Cells were passaged every 3 to 4 days by diluting cells to 2 × 105 cells/mL. Cell density and viability were measured using the trypan blue exclusion method on a Vi-cell XR cell viability analyzer (Beckman Coulter, CA).

Three separate transfections were performed using each promoter using the Nucleofector I system from Lonza (Cologne, Germany). 5 × 106 cells were transfected with 5 μg of linearized plasmids in each transfection. The transfected cells were transferred to 6-well tissue culture plates containing DMEM supplemented with 800 μg/mL of G418 (Sigma-Aldrich) for selection 24 h after transfection. Fluorescence-activated cell sorting (FACS) analysis was also performed using FACS Calibur (Becton Dickinson, Franklin Lakes, NJ) to determine the transient expression obtained using each promoter. Upon recovery of the stably transfected pools, at least nine clones were randomly selected from each stable pool by limiting dilution for a total of 30 clones to be carried forward for stability tracking."

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Eukaryotic cells - CHO EGFP using CpGrich from Yuansheng Yang, Bioprocessing Technology Institute, Agency for S.

Manufacturer protocol

Download the product protocol from Yuansheng Yang, Bioprocessing Technology Institute, Agency for S for CpGrich below.

Videos

Check out videos that might be relevant for performing Protein Expression Eukaryotic cells - CHO EGFP using CpGrich from Yuansheng Yang, Bioprocessing Technology Institute, Agency for S. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.