Wash Medium

RPMI 1640 (500ml) add Pen/Strep/Glutamine/Fungizone

ß-mercaptoethanol to 10 µM (90ul of Gibco 5.5x10-2M)

Sodium Pyruvate

HEPES Buffer to 12mM (6ml of 1M Stock, pH 7.0)

HAT MEDIUM:

250 ml Wash Medium: Add HAT supplement and 20% FCS

PEG 33% in RPMI 1640 (no additives) at 45°C (Sigma P-7777)

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Fill 5 96-Well Tissue Culture plates with 100µl/well HAT Medium and incubate 37°C

Myeloma Cells (3-4 T-75 Flasks with 6531 cells) Wash 2x with wash medium and resuspend in 50 ml tube with 10 ml wash medium: Incubate 37'C

Bleed mouse for +control then kill by cervical dislocation. Immerse mouse in 70% ethanol for 2-3 minutes, then aseptically remove spleen.

Push spleen through Cellector screen with 10ml syringe plunger. Further dissociate cells by drawing up and down in 5ml pipette 2-3 times.

Wash Spleen cells 2x with 10 ml wash medium. After second wash, resuspend in 10 ml wash medium.

Mix Myeloma and Spleen Cells in 50ml tube. Centrifuge for 5-10 min. at setting 6 on clinical centrifuge. At end of spin, take PEG out of 45°water bath.

Remove supernatant, leaving a semi-dry pellet.

To cell pellet, add 400-500 µl of PEG and gently mix cells with Pasteur pipette by drawing up and down no more than 2-3 times.

Incubate cell in 37°C water bath for 5 minutes.

Centrifuge at setting 4 for 3.5 min. Allow rotor to come to a complete stop.

Remove excess PEG

Resuspend in 10ml HAT medium GENTLY. Dilute to 50 ml HAT medium, and gently add 100ul per well to the 96-well plates.

DO NOT DISTURB CELLS FOR 3-5 DAYS!!. Don't look at them, don't touch them, don't move them. Leave them alone.

Feed HAT medium every 2-3 days, by removing then adding 100µl to each well.

When colonies become approx. 2/3 confluent, transfer into 12 well plate.

After 20 days, take cells off HAT medium, and feed RPMI-1640 complete.