Cultivation of hybridoma cells

Transcription

1 Monoclonal Antibody Facility Northwestern University Robert H. Lurie Comprehensive Cancer Center 710 N. Fairbanks Ct. Olson, Room 8370 Chicago, Illinois Izolda Popova Associate Director/Operations Manager Phone: Fax: Cultivation of hybridoma cells 1. Thaw frozen at -70 C or lower temperature suspension cells. Place a vial immediately to water bath at 37 C for one minute until small piece of ice is left inside. 2. Before plating wash out DMSO. Transfer thawed cells in 10 ml of cell culture medium in 15-ml blue cap sterile tube, spin down for 5 min at +4 C at 1000 rpm, discard supernatant and gently resus pend the pellet in 10 ml of fresh DMEM/F12 medium supplemented with 10-15% FBS. 3. Plate the cell suspension to 100 mm cell culture dish (BD, Falcon 100 x 15 mm style, cat # ). Optimal plating cell density is 1-3 x 106, the viability - 90% or higher. To check the cell viability mix 50 µl of cell suspension with 50 µl of 0.2% trypan blue in PBS and calculate number of stained (died) cell to the total cell number in a hemacytometer. 4. After two-three days of culture count the cells number and split the cells into 3-4 fresh culture dishes. Hybridoma cells can be removed from the plate by gentle pipetting and do not need to be trypsinized. 5. Repeat the splitting procedure several times until number of plates will reach the desired levels dishes are required to get finally 2 mg of the purified antibody (concentration of mab in hybridoma culture supernatant is µg/ml). 6. To freeze the hybridoma cells collect exponentially growing cells at >90% viability into 50-ml disposable sterile conical tube, spin down the cells for 5 min at +4 C at 1000 rpm. Discard supernatant, resuspend cells in freshly made, sterile freezing medium (FBS with 10% DMSO), aliquot 1-2 ml samples in cryogenic vials, place the vials into foam box and keep them at -20C freezer for 1 hour, and then place into 70 C freezer. In a few day transfer vials to liquid nitrogen for time indefinite. 7. To grow hybridoma cells for antibody production cultivate cells until the medium is yellow and most of the cells are died. Collect suspension in ml centrifuge containers, spin down at +4 C for 30 min with maximum speed, transfer the supernatants to fresh tubes and repeat spinning one more time. Use the supernatants to perform protein A, G, or L-affinity chromatography. For better results in affinity purification grow cells in serum free medium. 1

2 Standard Fusion Protocol 1. Grow myeloma cells few days prior fusion date. Myeloma cells culture should be maintained at <1x 10^6/ml density. Split myeloma cells the day before fusion to have next day 1x 10^7 cells (mouse splenocytes are fused with murine myeloma cell line at ratio 5:1); 2. Aseptically remove mouse spleen, place into sterile 100-mm cell culture plate containing 10 ml of sterile serum free DMEM/F12. Holding the spleen by sterile forceps perform spleen perfusion with serum free DMEM/F12 through 21g syringe needle. Pass splenocytes suspension through sterile Falcon 0.70 µm cell filter and resuspend cells into 15 ml tube. Centrifuge for 5 min and wash splenocytes two times in serum free DMEM/F12. Count cells; 3. Concurrently with the splenocytes wash myeloma cells twice with serum free pre-warmed DMEM/ F12 and centrifuge for 5 min at 800 rpm remove all traces of fetal calf serum (FCS); Count cells; 4. Add an appropriate number of myeloma cells to half volume of spleen cells and centrifuge together for 5 min at 1000rpm. (Immediately freeze down the remaining splenocytes); 5. Remove all supernatant with Pasteur and suspend cells. Gently disrupt the pellet by tapping the bottom of the tube. Place the tube in a 37 C water bath and keep it there during fusion. Pre-warm 50% PEG1500 to 37 ; 6. Add 1ml of warmed PEG 1500(w/v) to the pallet drop by drop over a period 1 minute while tapping the side of the tube to achieve thorough mixing. Continue to mix over next 1 minute at 37 C; 7. Dilute the PEG/cell mixture gradually as described below, continually but gently swirling the tube: 1ml serum free DMEM/F12 for sec 4ml serum free DMEM/F12 for 3 4 min 20 ml serum free DMEM/F12 at once 20 ml DMEM/F12+20%FBS at once 8. Centrifuge fused cells for 5 min at 800rpm. Remove supernatant and gently resuspend the cells. Mix with the appropriate volume of DMEM/F12 supplemented with 20% FBS and HAT selection medium and plate 120 µl of cell suspension to six 96-well plates. 9. Fusion plates are examined visually at after fusion for any abnormalities (i.e. contamina tion). At day 7 HAT selection medium have to be removed and cells are fed with fresh medium. After that cultures are examined every other day and fed. Once a majority of wells appear 50% confluent for growth the fusion products are screened by ELISA. 2

3 Indirect Standard ELISA Protocol I. Apply Antigen 1. Prepare an antigen solution in 0.05M Sodium carbonate buffer, ph 9.6* (1-10 µl /ml depending on antigen nature). Calculate total volume sufficient for coating all assay plates. Coat each well with 100µl of antigen solution. Cover the plate with adhesive film and incubate either at +4 C overnight or at RT for 2 hours (alternatively at 37 C for 1 hour) with shaking plates on a rocking platform at least for 30 min at moderate speed. 2. Wash the plates 3 times with PBS-T* using an automatic washer, or empty plates by shaking coating solution out of wells and filling wells with 200µl of PBS-T three times. As a final step, tap plates on paper towels to remove excess buffer. II. Block plate 1. Block wells by adding 200µl of blocking solution (0.25% Gelatin in PBS-T), seal and incubate for 1 hour at RT on rocking platform. 2. Wash the plates 3 times with PBS-T, as in Section I, step 2, of this protocol. III. React Primary Antibody 1. Add 100µl diluted with blocking solution primary antibody (hybridoma culture supernatant can be diluted 1:3) to each well, seal plates and incubate for 1 hour at RT at rocking platform. 2. Wash plates 3 times with PBS-T, as in Section I, step 2, of this protocol. IV. Apply Secondary Antibody 1. Dilute secondary anti-mouse antibody with blocking solution at dilution as recommended by manufacturer. Add 100µl per each well, seal and incubate for 1 hour at RT on rocking platform. 2. Wash plates 3 times with PBS-T, as in Section I, step 2, of this protocol. V. Add chromogenic substrate and develop 1. Prepare TMB* solution in 0.1M Sodium acetate buffer, ph 5.2* and filter through 0.45µm filter. Add 30% hydrogen peroxide to final concentration of 0.01% acetate. Immediately add 100µl per each well and allow to develop at RT for 5, 15, 30 min. 2. If desired, after sufficient color development add 50µl of stop solution, 10% (v/v) phosphoric acid. 3. Read plates with a plate reader. 3

4 Standard Fusion Protocol: Solutions Coating Buffer: 0.05M Sodium Carbonate buffer, ph g Na2CO g NaHCO g NaN3 Adjust to 1L by dh2o. Keep at +4 C PBS-T: Phosphate Buffered Saline, ph 7.4 containing 0.05% Tween-20 3M Sodium Acetate, ph 5.2: 408.3g of Sodium Acetate. 3H2O dissolve in 800ml water, adjust ph to 5.2 with glacial acetic acid. Adjust volume to 1L, autoclave. TMB 3,3, 5,5 -tetramethylbenzidine: Stock solution -100mg TMB dissolve in 10ml of DMSO. Aliquot and store in dark vials at +4 C For developing mix ex tempore 100µl of TMB and 9.9 ml of 0.1M Sodium acetate 4

5 Western Blot Protocol 1. Perform SDS-PAGE on samples to be analyzed. 2. Transfer proteins to Nitrocellulose or PVDF membrane according to the instructions provided by the manufacturer of the transfer apparatus. 3. Stain the blot using Ponceau S for 1-2 minutes to access the transfer of proteins onto the nitrocel lulose membrane. Rinse the blot in water to remove excess stain and mark the lane locations and locations of molecular weight markers using a pencil. 4. Rinse the blot in TBS-T for approximately 5 minutes. 5. Block the membrane using 5% non-fat dry milk in TBS-T for 1 hour at room temperature up to overnight at 4 C. 6. Dilute the primary antibody in blocking buffer and incubate 1-3 hours at room temperature on a rocking platform. 7. Wash the membrane in TBS-T x3 for 5 minutes and x2 for 8 min on rocking platform and apply the diluted conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature. 8. Wash the blot as in step 7 of this protocol. 9. Apply the detection reagent of choice in accordance with the manufacturers instructions. *The above information is only intended as a guide. The researchers should determine what protocol best meets their needs. Please follow safe laboratory procedures. 5