PREPARATION OF HYBRIDOMAS

Hazards involved: Aminopterin, Dimethyl Sulfoxide, PEG

See appropriate hazard sheet before proceeding.

Key reference: Galfre and Milstein (1981) for a detailed discussion of all methods.

Materials

Culture medium

RPMI 1640, Gibco cat. no. 21875.034

Advanced RPMI 1640, Gibco cat. no. 12633.012

For general tissue culture we use Invitrogen (Gibco) RPMI 1640 (500 ml.) cat. no. 21875.034. It is sound practice to test the culture characteristics of the medium prior to use.

We prefer Invitrogen Advanced RPMI 1640 (500 ml.) cat. no. 12633-012 for hybridoma preparations and cloning by limiting dilution.

To 450 ml. Advanced RPMI 1640, supplement with 10 ml. 100x L.Glutamine, Gibco cat. no. 25030.024; 5 ml. 100x Penicillin/Streptomycin antibiotics, Gibco cat. no. 15070.063; 176µl of a 1/100 diltn. of 2ME to give 50µM final; 50 ml. FCS,10% final.

For cell fusion culture medium, take 45 ml. complete culture medium above and supplement with 5 ml. FCS and 0.2 ml 250x Cholesterol, Gibco cat no 12531-018. Sterile filter 0.2 micron before use. We use the Advanced RPMI 1640 supplemented with 20% FCS and 1x Cholesterol, complete culture medium because the NS1 cell line is defective in Cholesterol metabolism. NS1 cells will grow well in this culture medium supplemented with only 1 to 2% FCS.

Other supplements for use in tissue culture:

Gibco, MEM NEAA (Non-essential amino acids) 100X cat. no. 11140.035

Sigma, Penicillin/Streptomycin/Neomycin 100X cat. no. P4083

Gibco, Gentamycin 100X cat. no. 15710.049

Gibco, GlutaMAX I 100X cat. no. 35050.038 GlutaMAX I is for general use, and can directly replace L.Glutamine in culture media.A second formulation GlutaMAX II may increase monoclonal antibody production from hybridomas.

Gibco, HAT 100x cat. no. 21060.017

Gibco, HT 100x cat. no. 41065.012

Gibco, Cholesterol 250x cat. no. 12531.018

Prepare sterile 2-Mercaptoethanol 100X, Sigma cat. no. M6250 (0.1ml/10ml. water) 87µl/500ml. culture medium (25µM final)

Other culture media for use in tissue culture:

DMEM, Gibco cat. no. 41966.029

Advanced DMEM/F12, Gibco cat. no. 12634.010

MEM, Gibco cat. no. 21090.022

IMDM (Iscoves' medium) 21980.032

Serum-free culture media:

OptiMEM, Gibco cat. no. 31985.047 A modification of Eagle's MEM buffered with HEPES and Sodium Bicarbonate,

X-VIVO 10 Cambrex (BioWhittaker) cat. no. 04.380Q 1 litre.

X-VIVO 15 Cambrex (BioWhittaker) cat. no. 04.418Q 1 litre.

X-VIVO 20 Cambrex (BioWhittaker) cat. no. 04.448Q 1 litre.

HL-1 Cambrex (BioWhittaker) cat. no. 344017. 500ml. Serum-free for hybridoma culture.

Hybridoma-SFM Gibco cat. no. 12045.084. 500ml. Serum-free for hybridoma culture.

Foetal calf serum

Gibco US FCS (500ml.) cat no. 16000.044

We prefer to purchase serum of North American origin to facilitate the shipment of hynridomas through US Customs. Gibco foetal calf serum is screened for virus and mycoplasma.

The FCS is heat-treated to destroy complement: 56^oC for 30 min. 100 ml aliquots are stored frozen until required.

Working Culture Medium RPMI 1640. 10% FCS

Prepare culture medium as required: Supplement 450ml. RPMI 1640 with 50ml. FCS (10% final); 5ml.100x L. Glutamine; 5ml. 100x Penicillin/Streptomycin antibiotics; 176µl (of a 1/100 diltn.) 2-Mercaptoethanol (50µM final). Filter the supplements into medium using a 50ml syringe and sterile 0.2µm bottle-top single use filter. Store 4 deg. C.

Add other supplements as desired. The medium is stored sterile at 4^oC and is used within two months. The complete medium is tested for sterility, and the ability to support growth of the NS1 Mouse myeloma line prior to use. The NS1 cell line is defective in Cholesterol biosynthesis and Gibco Cholesterol supplement may be added.

Additions of HAT or HT, are made as desired.

HAT:- 100x preparation, Littlefield formulation.

The Littlefield formulation for HAT contains Glycine which is essential for purine nucleotide biosynthesis.

From powder.

Cat. no. Final conc. in 100x conc. per 250 ml.

medium

Hypoxanthine Sigma H.9377 1 x 10^-4M 350mg

Aminopterin Sigma A.2255 4 x 10^-7 M 4.4mg

Thymidine Sigma T.9250 1.6 x 10^-5 M 96.75mg

L.Glycine BDH 28458 3 x 10^-6 M 5.5mg

Sodium Pyruvate BDH 44094 1 x 10^-3 M 2.75g

To approximately 100 ml. Hypoxanthine solution add 5M NaOH drop-wise with stirring until the Hypoxanthine is completely dissolved. Do not add more NaOH than is necessary. Add the remaining compounds (listed above) and make up to 250 ml. Filter sterilise through a 0.2 µ filter and store 50 ml aliquots at 4^oC. The preparation is tested for microbiological contamination before use.

GlutaMAX formulations: (there are two) are di-peptides, are cleaved within cells by an aminopeptidase, releasing L.Glutamine from the di-peptide with either L.Alanine (GlutaMAX I), or Glycine (GlutaMAX II). Supplement Gibco HAT with GlutaMAX II to provide Glycine for hybridoma preparation.

Supplements for bio-reactor culture:

Prepare sterile D.Glucose (25g/100ml water) 5ml./500ml. RPMI 1640 to give high Glucose 4.5g/L D.Glucose as in DMEM medium.

Prepare sterile Peptone, Sigma, cat no. P.5905, (10g/100ml. water) 10ml./500ml culture medium (0.2%)

GKN saline solution

An extremely useful Isotonic saline useful for washing cell preparations, is given by Fazekas de Saint Groth.

NaCl 8g, KCl 0.4g, Na₂HPO₄12H₂O 3.56g, NaH₂PO₄2H₂O 0.78g, Glucose 2g, Phenol Red 0.01g.

Make up to 1 litre with deionised distilled water and autoclave 121 deg.C (10 min.) to sterilise. The solution may be stored at room temperature.

Other saline solutions for use in tissue culture:

Dulbecco's Phosphate Buffered Saline (PBS) Sigma cat. no. D8537. No Phenol Red.

Hank's Saline (HBSS), No Calcium and Magnesium. Gibco cat. no. 14175.053

50% Polyethylene glycol

This laboratory has successfully used both BDH PEG 1500 and Merk PEG 4000. Autoclave 10g PEG to melt and allow to cool to approximately 40^oC and add 10 ml sterile GKN saline and mix well. The pH should be approximately 8.0 The solution is stored at room temperature. For the fusion the PEG solution is prepared freshly.

We now prefer to purchase PEG 1500 Boehringer (Roche) cat. no. 783.641 which is sterile and ready to use.

Cell culture cryo-preservation (Freezing) medium.

Dimethyl Sulphoxide. Sigma cat. no. D2650. Prepare freezing medium: 90ml. FCS + 10ml. Dimethyl Sulfoxide. DMSO is so toxic that it is sterile by itself. However, filter sterilise the freezing medium through a 0.2µ filter. Store at 4 deg. C. Use Nunc cryo-tubes (1.8ml) cat. no. 375418

Myeloma cell lines: Fusion partners for hybridoma preparation.

The cloned HAT-sensitive myeloma cell lines are cultured in RPMI 1640 + 10% FCS (50ml.) in still suspension culture in 80 cm² tissue culture quality plastic bottles, gassed with 5% CO₂/95% air at 37^oC.

Mouse lines (Balb/C)

NS1/1. Ag 4.1 Kohler and Milstein, 1976

NSO/u Clark and Milstein, 1981

X63/Ag 8.653 Kearney et al., 1979

SP2/0 Ag14 Sanchez-Madrid, 1983; Bluestone, 1987

The SP2/0 or X63/Ag 8.653 cell lines are favoured by U.S. laboratories because the hybridomas produced may be cultured in protein-free media. SP2/0 may be used to produce Hamster hybridomas. Hybridomas prepared using NS1 secrete a myeloma kappa light-chain as well as the hybridoma immunoglobulin; with SP2/0, NSO and X63/Ag 8.65 no myeloma kappa light-chain is produced.

Rat Lines (Lou rat)

Y3 (210.RCY3.Ag 1.2.3) Galfre et al (1979)

Hybridomas prepared from Y3 also secrete myeloma kappa chains. However, a non-secreting rat cell line YB213.0Ag3 (Y0) is available from J. Jarvis, MRC, Laboratory of Molecular Biology, Cambridge, UK.

We have also produced rat hybridomas from fusions using the mouse lines, NS1, NSO and X63/Ag 8.653.

Optimal growth of the various myeloma cell lines is obtained within the range 5 x 10⁴ cells/ml to 1 x 10⁶ cells/ml.

The cell lines are maintained in logarithmic growth phase in RPMI 1640 + 10% FCS culture medium. Some laboratories prefer to use IMDM (Iscoves') + 10% FCS culture medium which is more strongly buffered than RPMI 1640. Do not make fusions with cells which have been cultured for long periods of time. Periodically test the cell lines for HAT sensitivity and return to frozen stocks. It is important that the lines are periodically re-selected for HAT sensitivity by treatment with 6- Thio-Guanine cat. no. Aldrich A7.690.7 and then 8-Aza-Guanine cat. no. Sigma A 5284 to ensure cells are HGPRT negative and Thymidine Kinase negative.

It is essential that the cells are in good condition for successful fusions: diluting the cells to 2 x 10⁵ ml the day before the fusion ensures that the cell line is in the best possible condition prior to the fusion. It is sound practice to set up more than one flask of the cell line chosen to guard against accidental microbiological contamination.

0.1% Trypan Blue.

0.1% Trypan Blue, cat. no. BDH 34078, (100ml.) is prepared in PBS, filtered 0.45µm. to remove undissolved solid, and Sodium Azide (10mM final) added. Aliquots of cells are counted, either 1/2 or 1/20 in Trypan Blue solution, using a haemocytometer counting chamber. The viability of the cells, judged by dye exclusion, is consistently above 95% for a cell line in good condition.

Other materials

Trypsin. Gibco cat. no. 15090.046

TrypLE Express (Trypsin-like) Gibco cat. no. 12604.013. Test this before use because it has been reported that several cell lines have failed in gene transfections following use of TryplE Express to remove cell lines attached to plastic culture flasks.

Tissue Culture Plastics

Flasks, (80cm²) Nunc cat. no.153732, or use Costar 3376, incorporates a 0.2µm vent cap. (25cm²) Nunc cat. no.163371, or use Corning 3056, incorporating a 0.2µm vent cap.

Falcon tubes, (50ml), Becton Dickinson cat. no. 352070; (16ml). Becton Dickinson cat. no. 252099

Petri dishes, (50mm deep form) Sterilin cat. no. 124; (100mm) Becton Dickinson cat. no. 351005

Microtitre plates, 0.2ml., 96-well (micro-culture flat, bottomed wells) Becton Dickinson, cat. no.353072

0.2ml., 96-well (micro-culture U, bottomed wells) Becton Dickinson cat. no. 353077

Plates (2ml) 24-well (multi-well plate) Becton Dickinson 353047

Freezing ampoules (1.8ml) Nunc cryo-tubes cat. no. 375418

Immunization

For mouse x mouse hybridoma preparations. Immunisation schedules can vary depending on the antigen used, but usually Balb/c mice, 8-12 weeks old, are immunised as follows.

Two primary injections within one month. Subcutaneous + or - Complete Freund's Adjuvant with 10-50µg antigen (CFA/antigen 1:1 vol.). Use six mice. Then 8-10 days after the last immunizing injection, determine the serum antibody titre for each mouse using a RIA Trace Binding Assay, Williams et al. (1977) or ELISA.

N.B Current Home Office regulations only allow one immunization with CFA, the second immunization has to be done with IFA.

The minimum serum antibody titer of the immunized mice prior to hybridoma preparation ideally should be approximately 50% reactivity at a dilution of 1/2500. If the titre is low give further immunizations subcutaneously with incomplete Freund's adjuvant.

Within two months after the last immunization, the fusion priming injection of antigen, in PBS, is given I.V. 3-4 days later the cell fusion is performed.

The paper of Stähli et al. (1980) suggests an immunization schedule suitable for soluble antigens for the week preceding the fusion.

If the antigen consists of cells, immunizations of approximately 10⁸ cells in PBS are made I.P. and the final priming injection I.V.

In the past Gordon MacPherson has made intra-splenic immunizations for me for immunizations where the immunizing cell line or recombinant protein antigen is rapidly lost from the animal.

Transfection of Rat Basophilic cell line: For Rat x Rat hybridoma preparations a technique employed is to use the Rat Basophilic cell line (RBL) which has been stably transfected to express a recombinant cell surface molecule. RBL have the same MHC as AO rats, are trapped in the rat spleen, and only the recombinant antigen is reactive with the rat immune system.

Sp2.mIL6 is a transfected mouse B-cell line secreting mouse IL6, obtained from ATCC. Ref: Harris et al., 1992, J. Immunol. Methods 148:199-207) N.B. There are commercial restrictions if this cell line is used directly as a fusion partner for hybridoma preparation. However, it is permitted that the cell line is cultured and the rodent IL6 secreted into the tissue culture supernatant used as a supplement (20% final) in the culture medium used to produce and then re-clone both mouse and rat hybridomas.

Materials required

Hyper immunized Balb/C mouse boosted 3-4 days prior to the fusion. We prefer 4 days.

Myeloma cells in good condition e.g. 2 x 50ml flasks of NSI at 5 x 10⁵ cells/ml on the day of fusion will give a total of 5 x 10⁷.

Sterile petri dishes and sterile plastic tubes.

Sterile dissecting instruments under 70% ethanol.

Sterile Cell Strainers (100µm Nylon) Becton Dickinson cat. no. 352360

Centrifugations are made at room temperature. Beckman GPR (refrigerated) bench centrifuge of radius 12cm with swing out carriers.

Solutions required for the fusion

500 ml bottle of sterile complete culture medium Advanced RPMI 1640 + 20% FCS. The day after the fusion 100 ml of the complete culture medium containing 2 x HAT is required.

50% PEG solution at 37^oC

500 ml bottle of sterile GKN solution.

Trypan Blue counting solution.

Protocols

All operations are carried out at room temperature and avoiding delay.

Mouse IL6 supplement

Instead of using rodent thymocyte or spleenocyte feeders, for hybridoma preparation and subsequent re-cloning we supplement Advanced RPMI 20% FCS + Cholesterol with mouse IL6 TCS secreted by the SP2.mIL6 cell line. The SP2mIL6 cell line is grown in RPMI 1640 + 20% FCS medium for 4-5 days and the mouse IL6 TCS sterile filtered (0.2µm) to be used as a cell culture medium supplement.

Preparation of spleen cells

The spleen is removed aseptically from a hyper-immunized Balb/C mouse and gently teased apart using a pair of sterile forceps in 5 ml GKN solution. The spleenocyte cell suspension is passed through a sterile Cell Strainer (100µm) and washed through with a further 5 ml GKN solution. The cells are centrifuged for 5 min at 1000 rpm (100x g) and re- suspended in 5 ml GKN solution. The cells must be serum-free. A small aliquot is counted 1/20 with Trypan Blue solution. One will expect to obtain approximately 1 x 10⁸ spleenocytes from one mouse or 2-3 x 10⁸ spleenocytes from a rat spleen. We do not lyse the erythrocytes prior to the fusion. For a rat fusion only use half the cells from the spleen, the remainder may be cryo-preserved and used in the future.

Preparation of the myeloma cells

The myeloma fusion partner cell culture is counted 1/2 in Trypan Blue solution: A 50ml culture at a cell density of 5 x 10⁵/ml will give 2.5 x 10⁷ cells. The myeloma cells are centrifuged and re-suspended in 10 ml GKN solution, and repeated because the cells must be well washed to remove serum prior to cell fusion.

Mouse fusion protocol

Day -3 or 4

Priming antigen or cells to boost hyperimmune Mouse (I.V.)

Day 0

Fusion with polyethylene glycol (0.8 ml 50% PEG per 1x10⁸ spleen cells + 1x10⁷ NS1. Disperse into 5 x 96 well Titertek plates (0.2 ml/well) Advanced RPMI 20% FCS + Cholesterol + 20% Mouse IL6 TCS

Day 1

Take off 100µl medium from the cells and add 100µl Culture Medium + 2 x HAT in to the plates. Repeat every 3 days until the hybrid cells are growing well.

Day 10-14

Assay supernatents for interesting antibodies. Grow up and clone by limiting dilution into 0.2 ml x 96 well plates at 1 and 0.5 cells/well in Advanced RPMI 20% FCS + Cholesterol + 20% Mouse IL6 TCS. Cryo-preserve an appropriate number of cultures for further analysis.

Mouse hybridoma preparation: Mouse x Mouse: Ref: Galfre et al. (1977).

At 37^oC in the hood. Start timer and stir continuously.

0-1 min 0.8 ml PEG1500 (Boehringer)

1-2 min Stir for a further minute

2-3 min + 1 ml GKN

3-4 min + 1 ml GKN

4-5 min + 8 ml GKN

6 min + 40 ml GKN

The fusion is carried out at approximately 37^oC in a warm sterile microbiological safety cabinet. The cabinet may be warmed using a gas burner.

Mouse splenocytes (1 x 10⁸) are placed in a sterile Falcon tube with 1/10 the number of NS1 myeloma cells (1 x 10⁷) and a further 20ml GKN solution added. The cells are warmed at 37^oC for 5 min in a heated water bath and centrifuged for 5 min at 800 rpm (50x g). The supernatant is carefully removed by aspiration with a Pasteur pipette attached to a vacuum line. The Falcon tube is gently tapped to re-suspend the cell pellet and sterile 50% PEG solution at 37^oC (0.8 ml/10⁸ spleen cells) is added to the cell pellet over 1 min with gentle stirring using a 2ml plastic pipette. Continue to gently mix the cells with the pipette for a further 1 min so that the total exposure time to the 50% PEG solution is 2 min.

The PEG is diluted out by gently stirring in 1 ml GKN solution over 1 min, followed by a further 1 ml of GKN solution added over a further 1 min. 8 ml GKN solution is then added drop-wise with occasional stirring over 2-3 min, followed by the addition of GKN to 50 ml. Cell agglutination, particularly of the erythrocytes, should be evident.

The hybridoma preparation is centrifuged for five min at 800 rpm (50 x g) and the supernatant poured or aspirated off. The pellet is re-suspended by gently tapping the tube, and the cells and is re-suspended with approximately 85 ml Advanced RPMI 20% FCS + Cholesterol + 20% IL6 TCS and 0.2ml/well dispensed over four sterile flat bottomed 96-well Microtitre plates using an 8-channel pipette (sterilize by spraying with Klercide 70/30 sterile IPA and allow to dry in the safety cabinet) with sterile wide bore tips: Cell Saver Tips (Alpha cat. no. LW1018).

The plates are incubated overnight at 37^oC in a humidified incubator gassed with 5% CO₂/95% air. We favor taping round the plates with micro-porous tape (3M).

The following day remove 0.1ml from each well using a sterile 8-channel pipetter (with normal 0.2 ml sterile tips) and replace with 0.1 ml Advanced RPMI 20% FCS + Cholesterol + 20% IL6 TCS and containing 2 x HAT solution.

Three days later remove 0.1 ml medium and replace with 0.1 ml Advanced RPMI 20% FCS + Cholesterol + 20% IL6 TCS + 1x HAT solution and repeated again three days later after which time hybridomas should be evident within many of the wells. Periodically check the plates for microbiological contamination. The supernatants are screened on day 10-14 by trace binding assay or FACS analysis or by immuno-histology.

Rat fusion protocol

Day -3 or 4

Priming antigen or cells to boost hyperimmune Rat (I.V.)

Day 0

Fusion with polyethylene glycol (0.8 ml 50% PEG per 1x10⁸ spleen cells + 7x10⁷ Y3. Disperse into 4 x 24 well cell culture plates (1.0 ml/well) Advanced RPMI 20% FCS + Cholesterol + 20% Mouse IL6 TCS

Day 0 + 4 to 6 hours

Add 1.0 ml. Culture Medium + 2 x HAT in to the plates. Leave up to 2 weeks without changing the medium before assay.

Day 10-14

Assay supernatents for interesting antibodies. Grow up and clone by limiting dilution into 0.2 ml x 96 well plates at 1 and 0.5 cells/well in Advanced RPMI 20% FCS + Cholesterol + 20% Mouse IL6 TCS. Cryo-preserve an appropriate number of cultures for further analysis.

Rat hybridoma preparation: For Rat x Rat fusions we use the Y3 (Y3Ag1.2.3) rat myeloma. Rat spleenocytes(1 x 10⁸) and Y3 cells (5 x 10⁷). The fused hybridoma cells are re-suspended in culture medium IMDM (Iscoves') or Advanced RPMI medium containing 20% FCS + Cholesterol + 20% IL6 TCS. Plate 100ml fused cells over 4 x 24 well plates, 1 ml./well. After 4-6 hrs. add a further 1 ml./well of the complete culture medium supplemented with 2x HAT. Leave up to 2 weeks without changing the medium before assay. If you find that the high FCS or IL6 TCS interferes with your assay then, when the hybridomas are seen to be growing well, remove most of the culture medium from each well by aspiration and replace with approx. 1.5-2 ml. Gibco Serum-Free Medium (Hybridoma-SFM cat. no. 12045-084) or similar medium containing 1.5% FCS and leave for several days before assay. When you find positive clones, shift back into complete culture medium supplemented with 20% FCS, 20% IL6 TCS and 1x HT before re-cloning.

Day 27

Screen for antibodies. Re-clone:- Dilution clone at 1 or 0.5 cell/well containing feeder thymocytes in RPMI 20% FCS.

Day 40

Screen for antibodies. Bioreactor culture to obtain antibody for purification.

Positive Wells: After an interesting antibody is detected the goal is to clone the hybridoma as rapidly as possible. Early assaying is vital at each step. For positive mouse hybridoma wells, the cells are re-suspended using a sterile Pasteur pipette and are transferred to 1.5 ml

Advanced RPMI medium containing 20% FCS + Cholesterol + HT. (The hybridomas may be safeguarded by adding 0.2 ml of culture medium to fusion well.) Once the hybridomas have recovered clone the line by limiting dilution. It is sound practice to culture the hybridoma at an early stage for cryo-preservation, thus safeguarding against accidental microbiological contamination at a later stage.

Cloning: We favour the selection of positive clones by limiting dilution, 0.2 ml. Advanced RPMI 1640 + 20% FCS + 20% IL6 TCS + HT) at 100, 10, 1, 0.5 cells per well, 96 well FB culture plates. The 100 and 10/well plates are merely safeguards against contaminations and may be thrown out at a later stage)

The supernatants from the 1 and 0.5 cell/well plates are assayed when the clones are well grown, usually day 10-12. Several clones secreting antibody are grown up for cryo-preservation.

Positive clones are re-cloned at 1 and 0.5 cells/well and several final clones are cultured for cryo-preservation - pick the best for further use. It is sound practice to make test thawing of important hybridomas from cryo-preservation.

Production of antibody in animals No longer permitted in the UK and European laboratories.

Mice, Balb/c x DBA/2, (D2CF1) (Or appropriate rat strain.) are injected I.P. with 0.5 ml pristane, one week later with a cloned hybridoma line at 5 x 10⁶/0.5 ml PBS.

I favour re-cloning the new hybridoma after a single passage through mice as an ascitic tumour. Positive clones are selected and cultured for cryo-preservation and if required re-grown as an ascitic tumour in a large number of pristane-primed mice: e.g. 30 mice gives approximately 100 ml ascites fluid. Tissue culture supernatant will contain approximately 10µg/ml antibody and ascites approximately 5-10 mg/ml antibody.

Production of antibody from hybrids cultured in bio-reactors.

It is becoming less desirable to produce antibody from ascites and consequently antibody may be produced from high-density cultures of hybridomas in bio-reactors, the most commonly being the hollow fibre systems. Antibody concentration will be expected to be approximately 0.5 to 1mg/ml culture medium. After several weeks antibody may be extracted from the pooled TCS. We have used successfully the miniPERM from Heraeus Ltd. and Harvest Mouse from Serotec Ltd. We prefer to use the Integra Biosciences CL-1000 bioreactor flask.

Hamster hybridoma preparation: Specific antibody secreting hybrids have been obtained by Sanchez-Madrid using Syrian or Armenian hamsters. We have used the Syrian hamster fused with SP2/0 using the fusion protocol developed for the rat.

Freezing cells (Cryo-preservation)

5 x 10⁶ - 1 x 10⁷ cells in good condition, as judged by Trypan Blue exclusion, are centrifuged for 5 min at 1000 rpm (100 x g) and the pellet re suspended in 1 ml. sterile freezing solution (FCS containing 10% DMSO) at 4^oC) and placed within a sterile storage ampoule. The cells are frozen down on a holder within the neck of a liquid nitrogen Dewar flask according to the manufacturer's instructions, then transferred to storage in liquid nitrogen. An easier method of freezing the cell lines is to place ampoules containing hybrids in freezing medium directly into dry-ice (solid CO₂ pellets) for a minimum of one hour, then transferred to permanent storage in liquid Nitrogen. It is sound practice to freeze several vials for each clone.

Reviving the cell line: The frozen cultures are thawed quickly at 37^oC by placing the cryo-vials in a water bath to thaw and the cells are transferred to 30 ml complete medium at room temperature in a sterile 50 ml. Falcon tube with mixing. The cells are centrifuged for 5 min at 1000 rpm (100 x g) and the cell pellet re-suspended in 10 ml. Advanced RPMI 1640 20% FCS + Cholesterol in a 25 cm² and cultured, 37^oC, 5% CO₂/95% air, until well grown.

If the cells have been accidentally frozen down with a microbiological contamination the hybridoma may often be retrieved by limiting dilution culture with antibiotics. For a bacterial infection use Gentamicin, (bio-cidal) Gibco cat. no. 15710.049. For a fungal infection use Fungizone (bio-static) Gibco cat. no. 15290.018. It is no longer permitted to passage the hybridoma through animals as an ascitic tumour and re-selecting the cell line.

References.

Galfre,G., Howe,S., Milstein,C., Butcher,G.W. and Howard,J.C (1977) Nature 266, 550-552.

Galfre,G., Milstein,C. and Wright,B. (1975) Nature 277, 131-133.

Galfre,G and Milstein, C (1981) Methods in Enzymology 73, 3-46.

Kearney,J.F., Ardbruch,A., Liesegang,B. and Rajewsky, K.(1979) J.Immunol. 123 1548-1550.

Kohler,J. and Milstein,C. (1976) Eur. J. Immunol. 6, 511-519.

Lernardt,W., Andersson,J., Coutinho,A and Melchers,F.(1978) Exptl. Cell Res. 111, 309-316.

Littlefield,J.W. (1964) Science 145, 709-710.

Milstein,C. and Clarke,M.R.(1982) Somatic Cells Genetics 7, 657-666.

Stahli,C., Stäehelin,T., Miggiano,V., Schmidt,J. and Häring,P., (1980) J. Immunol. Methods 32, 297-304.

Williams,A.F., Galfre,G and Milstein,C. (1977) Cell 12 663-673.

Sanchez-Madrid et al 1983 J. Immunol. 130: 309-312

Bluestone 1987 PNAS 84: 1374

C.I.U. 2006, Michael J. Puklavec.