Lymphocyte Culture

1. RPMI 1640 culture medium (Gibco Invitrogen, Grand Island, NY) supplemented with 20% fetal bovine serum (FBS; Gibco Invitrogen). Stored at 4°C.

2. Gentamycin sulfate (Irvine Scientific, Santa Ana, CA) as a supplement to RPMI 1640 medium. Stored at 4°C and added to medium at 10 mg/mL.

3. Na heparin (Elkins-Sinn, Cherry Hill, NJ) as a supplement to RPMI 1640. Stored at 4°C and added to medium at 1000 U/mL.

4. L-Glutamine (Gibco Invitrogen) as a supplement to RPMI 1640. Stored at -20°C and added to medium at room temperature (4 mL/500 mL medium).

5. Phytohemagglutinin (PHA), lyophilized (Gibco Invitrogen). Stored at 4°C and reconstituted in distilled water at room temperature.

6. Potassium chloride and sodium citrate (Fisher Scientific, Pittsburgh, PA) for KCl-Na citrate hypotonic solution.

2.2. Collection of Cells and Preparation of Slides

1. Colcemid (Gibco Invitrogen). Purchased in lyophilized form and stored at 4°C. Used at room temperature after reconstitution in distilled water.

2. Methanol (Fisher Scientific).

3. Glacial acetic acid (Fisher Scientific).

4. Glass slides and cover glasses (Fisher Scientific).

2.3. Primed In Situ Hybridization

1. Programmable thermal cycler equipped with a flat plate for holding slides (MISHA, Shandon Lipshaw, Pittsburgh, PA) (see Note 1).

2. Dimethyl sulfoxide (DMSO; Sigma Genosys, St. Louis, MO) molecular biology or high-performance liquid chromatography (HPLC) grade.

3. Ethanol (Fisher Scientific).

4. PRINS primers (Research Genetics, Huntsville AL). The following oligonucleotide probes for the SRY gene (4) were HPLC purified and stored at -20°C (see Note 2):


The following probes for the SOX3 (SRY-related HMG box 3) gene (4) were HPLC purified and stored at -20°C:


5. Trinucleotides (dATP, and so on; TSA™ kit; Tyramide Signal Amplification, for Chromogenic and Fluorescence In Situ Hybridization and Immunohistochemistry, NEN Life Science Products, Boston, MA; see step 13).

6. Biotin-16-dUTP (Roche Molecular Systems, Alameda, CA; formerly Boehringer-Mannheim).

7. Potassium chloride (KCl).

8. Magnesium chloride (MgCl2).

9. Tris-HCl buffer (Sigma Genosys).

10. Bovine serum albumin (BSA; Invitrogen Life Technologies, Carlsbad, CA).

11. Taq DNA polymerase (Amplitaq, Perkin-Elmer, Foster City, CA) with TaqStart antibody (Clontech, Palo Alto, CA).

12. Formamide (Sigma Genosys) with NaCl and Na citrate: 20X SSC: 3 MNaCl, 0.3 M Na citrate, pH 7.0 (Invitrogen Life Technologies) to make formamide-SSC solution.

13. Tyramide Signal Amplification System (TSA™ Biotin System, NEN Life Science Products). The kit should be kept at 4°C until used, although the blocking reagent may be kept at room temperature. With proper storage, the kit is useful for 6 mo, after which the contents may become unstable.

14. Tween®20 (Sigma Genosys).

15. Triton X-100 (Sigma Genosys).

2.4. Fluorescence Microscopy: Visualization and Scoring

1. Fluorochrome-conjugated avidin (fluorescein isothiocyanate [FITC] or Texas red; Vysis, Downers Grove, IL).

2. Anti-fluorescein horseradish peroxidase (HRP; NEN).

3. Streptavidin-Texas red (NEN).

4. Counterstain: 4',6-diamino-phenylindole (Vysis).

5. Microscope equipped for fluorescence microscopy and an image capture system. An Olympus B-Max, U-M510 with triple bandpass filters, Red, Green and DAPI Bandpass, and Triple Bandpass Filter Set, DAPI/Green/Red (Vysis) was used here.

3. Methods

The basic method consists of four steps: (1) DNA denaturation, (2) annealing of primers, (3) chain extension in the presence of labeled nucleotides and Taq DNA polymerase, and (4) detection of the newly synthesized labeled DNA by use of antibodies complexed to fluorescent dyes.

3.1. An Overview of the General Procedures

1. A typical PRINS reaction mixture has a volume of 50 ^L and contains: 200 to 500 pmol of primer, 0.2 mM dATP, dCTP, and dGTP, 0.02 mM dTTP, 0.02 mMbio-16-dUTP, digoxygenin-11-dUTP, or fluorescein dUTP, 50 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.01% BSA, and 2 Us Taq polymerase (see Note 3).

2. The reaction mixture is placed on the denatured chromosome preparation.

3. Denaturation is accomplished by immersing slides in 70% formamide-2X SSC for 2 to 3 min at 72°C and then dehydrating them through a series of cold ethanol washes. After the slides are air-dried, the reaction mixtures are layered onto the slides and covered with cover glasses that are sealed in place with rubber cement.

4. The slides are put on a flat plate programmable thermal cycler and maintained at annealing temperatures for 5 to 10 min followed by extension for 20 to 30 min at 70 to 72°C. The reaction is stopped by immersing the slides in 500 mM NaCl/50 mM EDTA, pH 8, for 5 min at 72°C.

5. Labeled sequences are detected by immunocytochemistry and fluorescence microscopy.

6. Biotin-complexed fragments can be visualized by the use of fluoresceinated avidin and digoxigenin by the use of fluoresceinated digoxigenin antibody. The chromosome preparations are counterstained with 4,6-diamidino-2-phenylindole (DAPI) or propidium iodide and scored under ultraviolet (UV) light.

7. Blood lymphocytes, a ready source of DNA for PRINS testing, can be induced to divide in culture by addition of PHA. This allows preparation of mitotic spreads on glass microscope slides for evaluation by PRINS and other procedures, such as FISH). For these procedures, whole venous blood is drawn into heparinized glass tubes. The detailed procedure for lymphocyte culture in advance of PRINS is given in Subheadings 3.2 to 3.6. below.

3.2. Preparing the Culture Medium

1. Add 100 mL FBS to 400 ml RPMI 1640 in a separate container. Mix well. Store at 4°C and prepare fresh mixture every 30 d.

2. Into each bottle of medium add: 3 mL of Na heparin, 2 mL of penicillin-streptomycin, 2 mL of gentamycin sulfate, and 4 mL of L-glutamine.

3.3. Preparing the Phytohemagglutinin

1. Reconstitute one bottle of PHA with 10 mL of distilled water.

2. The PHA should be prepared fresh every 30 d and stored at 4°C.

3. Preparation of the KCl-Na citrate hypotonic mixture:

a. Add 0.560 g of KCl to 100 mL deionized or distilled water to make 0.075 M KCl solution.

b. Add 0.8 g Na citrate to 100 mL deionized or distilled water to make 0.8% solution, and mix the two solutions just before use: 2:1 (v:v), KCl/Na-citrate.

c. Store at room temperature and prepare a fresh mixture every 7 d.

3.4. Initiation of Lymphocyte Culture

1. Two cultures are initiated for each subject.

2. For each culture, add 10 mL of culture medium (see Subheading 3.1. above) to two 15-mL centrifuge tubes.

3. Using a Pasteur pipet, inoculate each centrifuge tube with 12 to 15 drops of whole blood. Each centrifuge tube represents a separate lymphocyte culture. Label the tubes.

4. Add 0.15 mL of PHA to each tube and mix well.

5. Incubate the cultures at 37°C for 72 h. (In general, the cultures may remain undisturbed for the full 3-d period, although some groups gently agitate the cultures several times each day.)

3.5. Harvesting the Lymphocytes

1. Using an 18-gage syringe needle, add 5 drops of reconstituted colcemid (10 ^g/mL) to each culture. The colcemid, which prevents formation of the mitotic spindle, thereby "freezing" the cells in metaphase, should be discarded after 1 mo of use.

2. After mixing well, incubate the cultures for an additional 60 to 120 min at 37°C in the colcemid.

3. Centrifuge at 250g for 10 min.

4. Remove the supernatant and break up the pellet by agitation with a vortex mixer.

5. Add 10 mL of hypotonic KCl-Na citrate mixture to each culture. Carefully resuspend the cells by gently inverting the centrifuge tubes manually. Allow the tubes to stand at room temperature for 30 min.

6. Gently resuspend the cells again by inverting the tubes. Then add 2 mL of freshly prepared (3:1) methanol/glacial acetic acid fixative directly to the contents of each tube. Mix the contents by inverting the tubes, and centrifuge the tubes again at 250g for 10 min.

7. Remove all the supernatant from each culture. Gently thump the tubes to break up the cellular pellets.

8. Add 10 mL of fresh fixative to each culture. Resuspend the cell pellets by inverting the tubes, and allow the cultures to stand at room temperature for 10 min, and centrifuge.

9. Repeat the preceding step.

10. The next steps are performed in a "harvesting room" with a humidifier. For maximal chromosome spreading, adjust the humidity to 55 to 65% (see Note 4).

11. Set a hot plate to 65°C, and check the temperature with a surface thermometer.

12. Centrifuge the cell suspension at 250g for 10 min.

13. Remove the supernatant from the centrifuge tubes, and add methanol/glacial acetic acid fixative to the pellet drop by drop until the suspension becomes semiclear. The amount of fixative required will depend on the size of the pellet. The final cell concentration may have to be adjusted after evaluation of the first slide.

3.6. Preparing Slides for PRINS

High-quality chromosome preparations are a key factor for obtaining satisfactory results after hybridization on glass slides.

1. Clean microscope slides are placed in a slide tray containing deionized water. The slides are chilled in a refrigerator.

2. One cold slide at a time is removed from the slide tray and maintained in a slanting position on a stand or a device (at about a 10°-20° angle).

3. Drop or place 40 to 50 ^L of cell suspension on the slide. Allow the cell suspension to roll down the slide. Wipe the back of each slide and shake off excess fixative and water. Label the slides, and place them on a hot plate at 65°C for 2 to 3 min.

4. Store slides at room temperature for 24 h after which they are ready for the PRINS procedure.

3.7. Primed In Situ Hybridization: Standard Protocol

1. Immerse slides in 0.02 N HCl for 20 min (see Note 5).

2. Now immerse slides in 70% formamide/SSC, pH 7.0, for 2 min at 72°C, to denature chromosomal DNA.

3. Dehydrate slides, by passage through a cold ethanol series, 70%-85%-100% EtOH, 5 min each. Air-dry.

4. Prepare reaction mixture in a final volume of 40 ^L containing: 50 pmol of each primer (see Note 6), 0.2 mM each dATP, dCTP, and dGTP, 0.02 mM dTTP, 0.02 mMbiotin/16-dUTP, 50 mM KCl, 10 mM Tris-HCl, pH 9.0, 2 mM MgCl2, 0.01%

BSA, and I U Taq DNA polymerase with TaqStart antibody (see Note 7). Pipet 40 ^L reaction mixture onto the freshly prepared slide (see Note 8).

5. Cover working area completely with a cover glass. Seal the ends of the cover glass in place with a thinly applied layer of rubber cement.

6. Incubate slides. Our incubations are carried out on a programmable thermal cycler equipped with a flat plate for slides (MISHA, Shandon Lipshaw; see Note 1). The program consists of one cycle of 10 min at annealing temperature (55-75°C) with an additional 30 min at 72°C for extension (see Note 9 for computation of annealing temperature).

7. After extension, the slides are removed from the cycler, the cover glasses are removed, and the slides are washed in 0.4X SSC at 72°C for 2 min to stop the reaction (see Note 10).

8. In our studies, biotin-labeled nucleotides are detected with the TSA™ Biotin System (see Subheading 3.8.).

3.8. Signal Amplification

1. Prepare Biotinyl Tyramide Stock Solution. Reconstitute biotinyl tyramide (amplification reagent) by addition of 0.3 to 1.2 mL DMSO (amount of DMSO will depend on the particular NEN kit that is used: 0.3 mL for 50-150 slides; 1.2 mL for 200-600 slides). Since DMSO freezes at 4°C, it is necessary to thaw the stock solution after removal from refrigerator.

2. For each test, dilute the stock solution 1:50 with 1X Amplification Diluent to prepare the working solution; 100 to 300 ^L of working solution are needed for each slide.

3. Prepare TNT washing buffer: 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20. The manufacturer advises that PBS may be used as an alternative buffer and that 0.3% Triton X-100 may be used instead of 0.05% Tween 20.

4. Prepare TNB blocking buffer: 0.1 M Tris-HCl, pH 7.5, 0.15 M. NaCl, and 0.5% Blocking Reagent (in kit). According to the manufacturer's protocol, the blocking reagent should be added slowly to the buffer in small amounts while stirring. The mixture should be heated to 60°C with continuous stirring (see Note 11). The blocking reagent should be dissolved completely (this may take several hours). After preparation, store at -20°C.

5. After hybridization, block slides by incubation with 100 to 300 ^L of TNB buffer. This may be done for 30 min in the water-filled chamber of the temperature cycler with biotin-labeled probes: 100 to 300 ^L SA-HRP (streptavidin-horseradish peroxidase: from the TSA kit) diluted 1:100 in TNB buffer. Fluorescein-labeled probes may be substituted: 100 to 300 ^L antifluorescein-HRP diluted 1:250 in TNB buffer. Optimal concentrations of HRP-labeled reagents should be determined for individual laboratories.

6. Wash slides in TNT buffer with agitation, 3 times for 5 min at room temperature.

7. Using a pipet, place 100 to 300 ^L working solution onto each slide. Maintain slides at room temperature for 5 to 10 min.

8. Repeat washing step (step 6).

3.9. Fluorescence Microscopy and Visualization

1. To each slide, add 100 to 300 ^L streptavidin-fluorophore conjugate diluted in TNB buffer; use dilution recommended by manufacturer (streptavidin-Texas red is used at 1:500).

2. Place slides in a humidified chamber for 30 min at room temperature.

3. Wash slides in TNT buffer with agitation, 3 times for 5 min at room temperature.

4. For background staining of chromosomes, place 2 drops DAPI II on slide and mount for microscopy. Blot excess DAPI II, add cover glass, gently seal ends of cover glass with rubber cement, and refrigerate at 4°C for 30 to 60 min (see Note 12).

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