RECOMMENDED QC PROTOCOL
M1 Bioassay
1. The M1 bioassay is performed using in vitro semi-solid agar cultures, which contain approximately 100 cells in 1 mL volumes of DME containing 20 % FCS in 0.3% agar.
2. Add 100 μL of sample or hLIF (10(E4) units/mL in 5% FCS in isotonic saline) in two-fold serial dilutions in duplicate to 35 mm petri dishes.
3. Add 100 μL of 5% FCS in isotonic saline to two control slides.
4. Incubate at 37°C in fully humidified atmosphere of 10% CO2 in air for 7 days.
5. Score the number of colonies that show differentiation (note: 50 units is defined as the amount of activity which results in 50% of the colonies being differentiated).
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Manufactured by CHEMICON International, Inc. LIF is protected under US Patent nos. 5,443,825, 5,750,654 and 6,261,548, European Patent no. 0285 448 and related foreign patents and is not available for resale.
Leukemia Inhibitory Factor (LIF) is a pleiotropic glycoprotein originally described to inhibit the proliferation of the murine myeloid leukemic cell line M1, while inducing differentiation into macrophages. Other activities were later identified to LIF, which is known by a variety of synonyms, including DIF, D-factor, DIA, DRF, CNDF, HILDA, HSF-III, and MLPLI. Human LIF exerts its actions through a receptor comprising a 190 kDa LIF-binding α-chain (130 kDa, mouse) and a 130 kDa signal-transducing β-chain (gp130), which is shared with CNTF, OSM, L-6 and IL-11. As such, it is a member of the gp130 family of the cytokine receptor superfamily. LIF receptors have been identified on several cells, including monocytes, liver, placenta and embryonic stem cells. Natural LIF is heavily glycosylated, showing an apparent molecular weight of 32 kDa to 62 kDa, depending on the source, but absence of glycosylation appears not to affect its biological activity. A single gene encodes LIF, which is secreted as a single chain glycoprotein containing 180 amino acids for human or mouse with a conserved disulfide bond. Human and mouse LIF share 78% sequence homology. Human LIF can activate mouse cells, but mouse LIF cannot activate human cells.
