ATM/CEP11

Product Name

ATM Gene Deletion Probe Detection Kit (Fluorescence In Situ Hybridization Method)

Product Introduction

This kit uses Orange Fluorescein labeled ATM probe and Green Fluorescein labeled CEP11, to combine ATM/CEP11 genes with the target site by in situ hybridization.

Product Main Components

The kit consists of ATM/CEP11 dual color probe as shown in Table 1.

Storage conditions & Validity

Keep sealed away from light at -20°C±5°C. The product is valid for 12 months. Avoid unnecessary repeated freezing and thawing that should not exceed 10 times. After opening, within 24 hours for short-term preservation, keep sealed at 2-8°C in dark. For long-term preservation after opening, keep the lid sealed at -20°C±5°C away from light.

Applicable Instruments

Fluorescence microscopy imaging systems, including fluorescence microscopy and filter sets suitable for DAPI (367/452), Green (495/517),and Orange (547/565).

Sample requirements

1. Applicable specimen type: fresh bone marrow specimen without fixation (preserved at 2-8°C for no more than 24 hours).
2. Take 1-3ml of heparin sodium anticoagulant bone marrow cells.
3. Sample preservation: after fixation, the cell suspension can be preserved at -20±5°C for no more than 12 months; the prepared cell slide can be preserved at -20±5°C for no more than 1 month. When the storage temperature of the sample is too high or too low, the cell suspension is volatilized excessively or polluted, the sample cannot be used for detection.

Test method

Related reagents

1. 20×SSC, pH 5.3±0.2
   Weigh 176g of sodium chloride and 88g of sodium citrate, dissolve in 800mL of deionized water, adjust the pH to 5.3±0.2 at room temperature, and complete to 1 L with deionized water. High-pressure steam sterilization, stored at 2-8°C, the solution shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
2. 2×SSC, pH 7.0±0.2
   Take 100mL of the above 20xSSC, dilute with 800mL deionized water, mix, adjust the pH to 7.0±0.2 at room temperature, complete to 1L with deionized water, stored at 2-8°C, the shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
3. Ethanol Solution: 70% ethanol, 85% ethanol
   Dilute 700ml, 850ml of ethanol with deionized water to 1L. The shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
4. 0.3% NP-40/0.4xSSC solution, pH 7.0-7.5
   Take 0.6mL NP-40 and 4mL 20×SSC, add 150mL deionized water, mix, adjust the pH to 7.0-7.5 at room temperature, with deionized water complete to a volume of 200mL. Stored at 2-8°C, the shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
5. Fixation solution (methanol: glacial acetic acid = 3:1)
   Prepare a ready to use fixation solution by mixing thoroughly 30ml of methanol and 10ml of glacial acetic acid.
6. 0.075M KCl solution
   Weigh 2.8g of potassium chloride, dissolve in 400mL of deionized water and complete to 500mL with deionized water. Stored at room temperature, the solution shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
7. Diamidinyl phenylindole (DAPI) counterstain
   Use commercially available anti-quenching DAPI counterstain.

Instructions

1. Sample pre-hybridization processing

• Sample collection: take 1-3ml of heparin sodium anticoagulant bone marrow cells.
• Cell harvesting: the uncultured marrow cells or the cultured marrow cell samples were aspirated to a 15mL centrifuged tube at the bottom of the tip, and centrifuged at 500g for 5min. The supernatant was carefully aspirated and discarded, leaving about 500μL of residual liquid to suspend the cells again.
• Cell washing: add 5ml of 1xPBS buffer solution, blow and mix up the heavy suspension cell precipitation, centrifugate 500g for 5min,carefully suck and discard the supernatant, and leave about 500μL of residual solution to heavy suspension cell; repeat once.
• Cell hypotonicity: add 10ml of hypotonic solution to each tube (37°C warm bath in advance), and water bath at 37°C hypotonic for 20min.
• Cell pre-fixation: add 1ml (10% volume) of fixed solution to the cell suspension after hypotonic treatment, gently blow and mix, centrifugate 500g immediately for 5min, remove the supernatant, and leave about 500μL of residual solution for cell suspension.
• Cell fixation: slowly add 10ml of the fixed solution to the cell suspension, leave it at room temperature for 10min to fix the cell, centrifugate 500g for 5min, and leave about 500μL of the residual solution to re suspend the cell; repeat once (or fix the cell several times until the cell is precipitated, washed and cleaned).
• Preparation of cell suspension: after the last centrifugation of cell fixation, the supernatant is sucked off, and a proper amount of fixed solution is added to make cell suspension with appropriate concentration.
• Preparation: take 10μL cell suspension drop to slide, aging at 56°C for 0.5h.

2. Slides pretreatment

• The slides were rinsed twice in 2xSSC solution at room temperature for 5min each time.
• Dehydration: the cell drops were placed in 70% ethanol, 85% ethanol and 100% ethanol for 2 minutes respectively and then dried naturally.

3. Denaturation and Hybridization
The following operations need to be carried out in the darkroom.

• Take out the probe, leave it at room temperature for 5min, turn it upside down with force, mix it well, and then centrifuge it for a short time (no vortex instrument vibration). Take 10μL of it and drop it into the cell drop hybridization area, immediately cover the cover glass of 22mm × 22mm. The probe should be evenly expanded under the cover glass without bubbles, and seal the edge with rubber glue (the edge must be completely sealed to prevent the dry piece from affecting the test results in the hybridization process).
• The cell drops were placed on the hybridizer and denatured at 88°C for 2 min (the hybridizer should be preheated to 88°C) and hybridized at 45°C for 2-16 h.

3. Washing
The following operations need to be carried out in the darkroom.

• Carefully remove the sealing glue around the cover glass with tweezers to avoid sticking or moving the cover glass, immerse the sample in 2xSSC for about 5S, take it out, gently push a corner of the cover glass to the edge of the slide with tweezers, and gently remove the cover glass with tweezers.
• Place the sample at 2xSSC room temperature for 1 min.
• Take out the sample and immerse it in 0.3%NP-40/0.4xSSC solution preheated at 68°C for 2min.
• Take out the sample and immerse it in deionized water preheated at 37oC in advance for 1min; dry it naturally in the dark place.

4. Complex dyeing
The following operations should be performed in a darkroom
10μl DAPI compound dye is dropped in the hybridization area of the glass slide and immediately covered. The suitable filter is selected for glass slide observation under the fluorescence microscope.

5. FISH results observation
Place the stained sections under a fluorescence microscope and the cells area is first confirmed under a low magnification objective (10×);under magnification objective (40×) a uniform cells distribution is observed; then the nucleus size uniformity, nuclear boundary integrity,DAPI staining uniformity, no nuclei overlapping, cells clear signal are observed in the high magnification objective (60x, 100x).

Common Signal Type Interpretation

ATM signal

CEP11 signal

Negative: 2 Orange ; 2 Green (2R ; 2G)

Positive: 1 Orange ; 2 Green (1R ; 2G)

Precautions

1. Please read this manual carefully before testing. The testing personnel shall receive professional technical training. The signal counting personnel must be able to observe and distinguish orange red and green signals.
2. When testing clinical samples, if it is difficult to count the hybridization signals and the samples are not enough to repeat the retest, the test will not provide any test results. If the amount of cells is insufficient for analysis, again, the test will not provide test results.
3. The formamide and DAPI counterstaining agent used in this experiment have potential toxicity or carcinogenicity, so they need to be operated in the fume hood and wear masks and gloves to avoid direct contact.
4. The results of this kit will be affected by various factors of the sample itself, but also limited by enzyme digestion time, hybridization temperature and time, operating environment and limitations of current molecular biology technology, which may lead to wrong results. The user must understand the potential errors and accuracy limitations that may exist in the detection process.
5. All chemicals are potentially dangerous. Avoid direct contact. Used kits are clinical wastes and should be properly disposed of.
6. This product is for clinical diagnosis and scientific research.