Herramientas o tecnologías para el desarrollo de la aplicación web

and HUC-IT ceHs

2-13-1 Aim and principle

A im : Q uantitative determ ination of h u m an stem cell factor (SCF) in the conditioned m edium .

P rin c ip le : M onoclonal antibody specific for SCF im m obilizes th e fac to r in th e co n d itio n ed m ed iu m . A seco n d enzym e-linked polyclonal antibody b in d s to the factor and a colour reaction is developed in proportion to the am ount of SCF bo u n d in the initial step.

2-13-2 Materials and m ethods

C onditioned m edium w as prepared as previously detailed [2-12-2] u sin g 7% FCS F12 m edium for b o th SV-HUC-1 an d SV-HUC-IT cells.

ELISA kits used were com m ercially available. [BIOTRAK, A m ersham U.K.], [Q uantikine. R&D system U.S.A.].

Procedure as p er m an u factu res instru ctio n s. Briefly, sam ples were added to th e te s t m icro titre w ells w hich w ere p reco ated w ith m onoclonal m urine antibody against SCF. Bound SCF w as m easured using polyclonal SCF antibody conjugated to horseradish peroxidase. A curve of optical d ensity v ersu s the co n cen tratio n of SCF w as generated using sta n d ard recom binant h u m an SCF. C oncentration of SCF in th e sam ple w as determ ined by com paring th e optical density of the sam ples to th is stan d ard curve. The assay range for h u m an serum SCF concentration is 31.3pg/m l to 2000pg/m l.

M & M 64

2-14 Murine thym ocyte proliferation assay

A im : The detection of interleukin one(ILl)-like activity. P rin cip le:

1. A subpopulation of thym ocytes (T lym phocytes) exposed to ILl express interleukin two (IL2) receptors.

2. A subpopulation of thym ocytes interact w ith a suboptim al levels of m itogen (Concanavalin A) and release IL2.

3. IL2 s tim u la te d IL 2 -re ce p to r e x p re ss in g cells u n d e rg o proliferation.

4. T hym ocyte pro liferatio n is detected by tritia te d thym idine incorporation, and is related to the level of ILl (Krakauer, T. et al

1982).

2-14-2 Materials and m ethods

P rep aratio n of m u rin e thym ocytes: Thym ocytes w ere h arv ested from the 2-3 w eeks old CBA/H mice w hich were killed by cervical spine dislocation.T he thoracic cavities were opened u n d e r sterile condition, th e th y m u s g lan d s situ a te d above the h e a rt w ere rem oved and a single cell su sp en sio n of thym ocytes w as produced in RPMl 1640 m edium [ICN] supplem ented w ith 10%(v/v) FCS, lO O u/m l benzyl penicillin, lOOpg/ml streptom ycin, an d 2mM L- glutam ine. C ellularities w ere determ ined w ith a C oulter counter

[ZM, C oulter Electronics, U.K.] and adjusted to lO ^cells/m l.

M itogen: C oncanavalin-A (ConA) will itself stim u late thym ocyte proliferation. A suboptim al concentration of ConA w hich will not itself stim u late thym ocyte proliferation w as determ ined (1 pg/m l) for use during the assay of ILl activity.

M & M 65 T ritiated thym idine: [m ethyl-®Hjthym idine ([®H]TdR) [Am ersham ] specific activity, 185G Bq/m m ol, w as diluted to a w orking solution of 300 K B q/m l w ith RPMI 1640 m edium co n tain in g 4 0IU /m l gentam icin.

100 pi of 10^ th y m o cy tes/m l an d 50 pi of 4 p g /m l ConA w ere added to a n u m b er of flat-bottom ed wells of a 96 well m icrotitre plate (Nunc, D enm ark) 50 pi of either conditioned m edium , or a range (2 - 100 U /m l) of reco m b in an t h u m an ILl a [Im m unex, U.S.A.] to produce an ILl titration curve, w as added to each well. The plate w as incubated for 3 days in a 37°C 5% COg in air, fully hum idified in cu b ato r after w hich 25 pi of 300 K B q/m l [®H]~TdR w as added to each well. Incubation w as continued for 16 hours. Cells w ere h arv ested u sin g a T itertek cell h a rv e ste r [Skatron, Norway] onto glassfiber filter paper [Titertek]. After thorough oven drying, individual filter discs were placed in scintillation vials and 2 m is of scintillant [O ptiphase 'Safe', FSA Laboratories, U.K.] added. [®H]-TdR u p tak e by proliferating cells w as determ ined u sin g a liquid scintillation counter[LKB1214, Rackbeta, U.K.]. Each vial was counted for a 5-m inute period and a count per m inute' (CPM) value determ ined. T his figure reflects the proliferation of thym ocytes stim ulated by ILl.

2-15 [^H] thym idine incorporation assay determ ining the