FTOC Protocol 小鼠胚胎胸腺培养

Protocol

Fetal Thymus Organ Culture

INTRODUCTION

The generation of functionally competent T-cells from their progenitors involves a series of developmental events including proliferation, differentiation, and survival. T-cell development is a non-cell-autonomous event, and requires interactions with thymic stromal cells. Fetal thymus organ cultures provide an in vitro system in which isolated embryonic thymus lobes can be maintained in culture, allowing the study of T-cell development as well as thymic stromal cell function. This system remains the only in vitro system that supports a complete program of T-cell development, including positive and negative selection of the developing T-cell receptor repertoire. Modifications of the basic fetal thymus organ culture system, such as hanging drop cultures and reaggregate thymus organ cultures, provide useful systems to analyze thymus colonization and thymic stromal cell function, respectively.

MATERIALS

Reagents

All media should be prewarmed to 37°C before use.

10% CO2 in air, contained in gas cylinder

2'-deoxyguanosine (dGuo) (Sigma-Aldrich, D0901)

Prepare a 9 mM stock in 1X PBS. It takes ~1 h at 37ºC for the dGuo to dissolve.

Mix the solution well during this period.Dilute the dGuo stock to a final concentration of 1.35 mM in1X PBS (see Step 3).

Dulbecco’s modified Eagle’s medium (DMEM) for thymus organ culture

70% ethanol

Mice, pregnant female (gestational age E14-E16)

1X PBS (Ca++/Mg++-free)

RF10-H medium

10X trypsin (Sigma-Aldrich,T4674)

Prepare a 1X trypsin solution by diluting the 10X stock in Ca++/Mg++-free PBS containing 0.02% EDTA.

Equipment

Artiwrap sponges, 1 cm2 (Medipost Ltd.)

Aspirator tube assembly (Sigma-Aldrich, A5177)

Boxes (rectangular, plastic) with fitted lids (Watkins and Doncaster,E6052)

Bunsen burner, fish-tail

Filters (isopore membrane) with 0.8-µm pore size (Millipore,ATTP01300)

Forceps, watchmaker, Dumont #5 (TAAB)

Incubator, preset to 37°C

Microcentrifuge

Microcentrifuge tubes, 1.5 mL

Micropipette, 1 mL

Microscope (stereo-dissecting) with magnification range 0.8X-5X(e.g., Zeiss, Stemi SV)

Petri dishes, 90-mm diameter, sterile bacteriological grade(Sterilin)

Plate inserts for multiwell plates (e.g., for a six-well plate,use Millicell 0.4-µm plate inserts; Millipore, PICM03050)(optional; see Step 2.i)

Plates, Terasaki (Sterilin)

Scissors, surgical

Tape

Tubing, glass, to make capillary pipettes (Fisher Scientific UK, FB51460)

Vortex mixer

METHOD