Paul.xu at 17:13, 2 January 2013

2013-01-02T17:13:19Z

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== Small Scale (6-well) ==

1. Use T75 cm2 or T225 cm2 cell culture flasks to grow the HEK 293T cells and split the cells every 2-3 days when cells are 100% confluent. 

2. Seed 2.5 x 105 HEK 293T cells per well in a 6-well plate and add 2 ml DMEM with 1X pen/strep and 10% (v/v) FBS, swirl the plate gently to ensure even cell dispersal in each well, and incubate overnight at 37 °C in a 5% CO2 humidified chamber. 

3. When HEK 293T cells reach 40% confluency, discard the media and add fresh 2 ml DMEM with 1X pen/strep and 10% (v/v) FBS to the wells. Perform transfection assays. 

4. Aliquot 90 μl serum-free DMEM into a sterile 1.5 ml microcentrifuge tube. Pipette 3 μl L2K into the serum-free DMEM and gently mix the tube (finger vortex). Incubate for 5 minutes at room temperature. 

5. Add 1 μg of MiniPrep purified plasmid DNA (DNA stock= 100 ng/μl) into DMEM-L2K mixture, finger vortex, and incubate for 15 minutes at room temperature. 

6. Pipette the transfection mixture dropwise onto HEK 293T cells, and swirl the 6-well plate gently to allow even distribution of the transfection mixture. Incubate the 6-well plate at 37 °C in a 5% CO2 humidified chamber. 

7. Add 1 ml fresh DMEM with 1X pen/strep and 10% (v/v) FBS to each well 24 hours post-transfection and incubate for another 48 hours (total 72 hours). 

8. Harvest 1 ml of supernatant from each well at three days post-transfection and microcentrifuge the samples at 16,000 g for 10 minutes at room temperature.  Samples can be stored at 4 °C. The length of storage at 4 °C is protein dependent. 

== Large Scale ==

Use 10-layer cell factories (6360 cm2 surface area) or smaller cell factories or T-flasks for protein production

 

1. Purify 1 mg of DNA for transfection using a MaxiPrep plasmid purification kit. A 500 ml overnight culture  should produce at least 1 mg of pure DNA. Check purity of DNA by measuring A260/A280 ratio; should be above 1.8.

 2. Scale up HEK 293T cells to 2.0 x 108 cells. Each T225 cm2 flask grown to 100% confluency contains and average of ~2.25 x 107 cells.

 3. Add 1.2 L DMEM with 5% (v/v) FBS to a 10-layer cell factory. Add 2.0 x 108 HEK 293T cells to the cell factory and distribute cells evenly to all layers of the vessel. It is very difficult to visualize the confluency of cells in the cell factory. As an alternative, set up a T75 cm2 flask with an appropriate number of cells, using the same cell number to surface area ratio as performed with the 10-layer vessel. Monitor this flask for growth rates. See the associated video for instruction on handling the cell factory. Incubate overnight at 37 °C with 5% CO2 to allow for cell attachment and growth. 

 

4. Perform large-scale transfection when adherent HEK 293T cells are 70% confluent. Prepare the PEI-DNA transfection mixture (3:1 w/w PEI to DNA ratio) in a biosafety cabinet using a sterile T75 cm2 flask. Mix 0.84 mg of DNA with 84 ml of sterile 1X PBS, then add 2.5 ml of PEI (2.5 mg total PEI). Incubate at room temperature for 15 minutes. Solution should become cloudy. 

 

5. Pour the PEI-DNA transfection mixture slowly into the cell factory and thoroughly distribute over all layers of the vessel. Optional: for increased expression yields, add valproic acid (4 mM final concentration). Incubate at 37 °C with 5% CO2 for four days. 

 

6. Harvest supernatant 4 days post-transfection. Centrifuge the conditioned media at 6000 x g for 30 minutes at 4 °C. Further filter the supernatant using a 0.22 μm Stericup vacuum filter apparatus. The 10-layer cell factory can be reused, seeBox 3 for cleaning instructions. It is key that cleaning is initiated immediately after supernatant harvest; do not let the cells dry onto the vessel surface. 

 

7. Concentrate the supernatant to 75 ml using the Centramate tangential flow filtration system. 

 

8. Add 500 ml of PBS and re-concentrate to 75 ml. Repeat three additional times to completely buffer exchange the sample. 

 

9. Equilibrate a 1 ml Ni or protein A column with 1X PBS and apply concentrated sample by gravity flow at a rate <1 ml/min. 

 

10. Wash the column with 30 ml of 1X PBS-Tween20. 

 

11. Elute protein using 250 mM imidazole in PBS for 6XHis tagged protein or low pH glycine solution to elute protein.

 

{| border="1"
|-
| Vessel
| Surface area
|-
| 6-well plate
| 9.5 cm2 (each well)
|-
| 100 mm dishes
| 55 cm2
|-
| 245 mm dishes
| 500 cm2
|-
| T75 cm2 flask
| 75 cm2
|-
| T175 cm2 flask
| 175 cm2
|-
| T225 cm2 flask
| 225 cm2
|-
| Roller bottle- regular
| 850 cm2
|-
| Roller bottle- expanded surface
| 1700 cm2
|-
| 1-layer CellSTACK
| 636 cm2
|-
| 2-layer CellSTACK
| 1272 cm2
|-
| 5-layer CellSTACK
| 3180 cm2
|-
| 10-layer CellSTACK
| 6360 cm2
|-
| 40-layer CellSTACK
| 25,440 cm2
|}

 

== Protocol 2 ==

1. Extract plasmid with chloroform to kill bacteria.

2. Add 1 mg DNA to 100 ml serum free DMEM.

3. Add 2 ml 1 mg/ml PEI 25K.

4. Incubate 10 min at RT.

5. Add to one HYPERFlask containing 450 ml DMEM + 2% FBS.--> Final is 1.6% FBS

Triple flasks: 150 μg DNA + 250 μl PEI in 30 ml serum free DMEM-->70 ml DMEM + 2% FBS

HYPERFlasks: 1 mg DNA + 2 ml PEI. (in 100 ml serum free DMEM)-->450 ml DMEM + 2% FBS

2125 cm2 expanded surface roller bottle (Greiner): 0.5 mg DNA + 50 ml serum-free DMEM, mixed, + 1 ml PEI stock (1 mg/ml), add to roller bottle containing 200 ml of DMEM, 2% FBS.

6. Collect conditioned medium 4–7 days after transfection.

7. Filter through a 500-mL filter unit with a 0.45- m membrane (Corning) and transfer the filtrate into two 500-mL centrifuge bottles (Corning).

If protein has 6His tag, Ni beads can be used for purification: dialyse filtered medium against 25 mM phosphate buffer saline (PBS, pH8.0) at 4 °C o/ before loading to Ni column.

8. Purify protein using Ni beads or protein A beads. 

[[Category:Protocols]]

293T Protein Production - Revision history

Paul.xu at 17:13, 2 January 2013