For each transfection, 40 pg of transfection vector will be needed. It is helpful to obtain a high yield of pure transfection vector in case the transfection needs to be repeated in the future. In addition, one confluent T75 flask of NS0 cells is required per transfection. NS0 cells must be grown in nonselective medium prior to transfection. (Subculture three times per wk by splitting 1:5 in fresh nonselective medium.) All cell handling should be performed under a sterile hood to prevent contamination.
3.4.1. Stable Transfection of NS0 Cells 1. Digest transfection vector as follows. Incubate 4-16 h at 37°C (see Note 7).
40 pg Transfection vector X pL
10X SaH buffer 10 pL
100X BSA 1 pL
dH2O X pL
Total volume 100 pL
2. Transfer entire digest volume to Gene Pulser cuvet. Keep on ice.
3. Prewarm 40 mL of nonselective medium at 37°C.
4. Obtain one confluent T75 tissue culture flask containing NS0 cells grown in non-selective medium.
5. Loosen NS0 cells by smacking flask against the palm of the hand.
6. Transfer entire volume of cells into a sterile 50-mL conical tube and centrifuge for 10 min at 300g with low brake.
7. Resuspend cell pellet in 5 mL of sterile PBS, then fill to 50 mL with sterile PBS. Repeat PBS wash.
8. Determine cell concentration using a hemacytometer.
9. Centrifuge cells again for 10 min at 300g with low-brake. Resuspend cell pellet in sterile PBS such that the cell concentration is 1.1 X 107 cells/mL (see Note 8).
10. Add 900 pL (or 1 X 107 cells) to the cuvet containing the digest. Gently mix cells by pipetting, being careful not to create excessive air bubbles.
11. Incubate on ice 5 min, then thoroughly dry metal sides of cuvet, which will come in contact with electrodes.
12. Place cuvet into electroporator and slide apparatus until there is contact between cuvet and electrodes. Adjust electroporator settings to 1.5 kV and 3.0 pF.
13. Pulse the cuvet by pressing the two red buttons. Repeat, then incubate cuvet on ice 5 min.
14. Pipet cuvet contents into prewarmed selective medium, invert several times to mix. Pour into a sterile reservoir, and with a multichannel pipettor, add 50 pL of cells per well to 8 sterile flat-bottom 96-well plates.
15. After 18-24 h, add 150 p,L of prewarmed selective medium to all wells.
16. Three days later, remove medium from each well with aspiration manifold and add 150 p,L of fresh selective medium to cells.
17. Incubate at 37°C for 3-4 wk (see Note 9), until colonies appear in wells and medium begins to turn yellow.
3.4.2. Screening Transfectants Using Fc-Specific ELISA
While preparing for the ELISA, make sure to have the appropriate positive and negative controls (see Note 10).
1. Coat plastic 96-well flexible plates with 100 p,L of coating buffer containing 2-5 ^g/mL of primary Ab (goat anti-mouse IgG and IgM). Cover plates with lid and incubate overnight at 4°C.
2. Wash plates three times with 200 p,L of PBST using a microplate strip washer. Invert plates and tap to get rid of residual wash buffer in wells.
3. Add 50 ^L of blocking buffer to all wells.
4. Add 50 p,L of medium (1:2 dilution) from transfectant wells that contain colonies. Be careful not to disturb colonies at bottom of wells when removing medium. Incubate at 37°C for 1 h.
6. Make a 1:10,000 dilution of secondary Ab (peroxidase-conjugated goat anti-mouse IgG) in blocking buffer, then add 100 ^L per well. Incubate at 37°C for 1 h.
8. Prepare OPD solution according to manufacturer's protocol; add 100 p,L of fresh OPD solution to all wells. Incubate at room temperature for 30 min in the dark, or until brown color develops.
9. Read plate using a multiwell plate reader set to 450 nm, with a reference wavelength of 630 nm. The highest-producing clones are those with the highest OD reading.
10. Expand the highest-producing clones to a 24-well plate using selective medium. Grow cells until confluent to prepare for subcloning.
3.4.3. Subcloning of Highest-Producing Transfectants
1. Count cells from 24-well plate using a hemacytometer.
2. Subclone each transfectant as follows: remove a volume containing approximately 96 cells, add to 15 mL of prewarmed selective medium, then aliquot 150 p,L per well in sterile 96-well plates, such that a 1 cell/well concentration is obtained (see Note 11).
3. Place plates in 37°C tissue culture incubator and allow to grow for 3-4 wk.
3.4.4. Screening Subclones Using ELISA Specific for the Protein Moiety
While the initial transfectants were screened with Fc-specific secondary Ab, it is important to subsequently screen subclones with an Ab specific for the protein moiety fused to murine Fc (see Note 12).
1. Coat flexible 96-well plates with 100 pL of coating buffer containing 2-5 pg/mL of primary Ab (goat anti-mouse IgG and IgM) to all wells. (One plate is needed for every 10 clones to be analyzed.) Cover plates with lid and incubate overnight at 4°C.
2. Wash plates three times with 200 pL of PBST using a microplate strip washer. Tap plates to get rid of excess wash buffer in wells.
3. Add 196 pL of blocking buffer to all wells in row A and 100 pL of blocking buffer to all remaining wells.
4. Remove 4 pL of supernatant from each subclone and add to well A1. Repeat for wells A2-A10 with supernatant from additional subclones. In well A11, add positive control protein of appropriate concentration. Add no medium to negative control well A12.
5. Using a multichannel pipettor, mix contents in row A and draw up 100 pL. Mix aliquots from row A with blocking buffer contained in row B. Continue with 1:2 dilutions for rows C-H (see Note 13).
6. Incubate plate at 37°C for 1 h. Wash plate three times with a microplate strip washer. Tap plate.
7. Dilute protein moiety-specific secondary Ab in blocking buffer at a ratio recommended by the manufacturer. Add 100 pL of secondary Ab to each well.
8. Incubate plate at 37°C for 1 h. Repeat wash.
9. Develop plate with OPD as in Subheading 3.4.2., step 8. Read plate as in Subheading 3.4.2., step 9.
10. Select the highest-producing clone (as determined by highest OD) for large-scale production. Freeze down four to five additional high-producing clones by resus-pending each in 2 mL freezing medium; store at -80°C.
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