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Preparation of Metaphases from Blood

Preparation of Slides (optional) • Store slides in HCl-EtOH (1% HCl in 70% ethanol) over night • Rinse slide for one hour under flowing water • Store in water at 2 to 8 °C until used

Chromosomal Staining Protocol • Prepare 0.5 ml Giemsa Stain Solution in 10 ml PBS (H15-002) • Preheat a mixture of 1 ml Trypsin (L11-001) per 10 ml PBS to 37 °C • Prepare three Slide Tray (PAA11400145) chambers: 1. prewarmed Trypsin/PBS 2. Aqua purificata grade 3. Giemsa Stain Solution/PBS • Dip a slide with fixed cells 2 to 3 times in chamber 1 (if the staining turns out to be to weak increase number of dipping) • Dip the slide twice in chamber 2 • ncubate the slide in chamber 3 for 5 – 10 minutes (start with 5 minutes and increase time if needed) • Rinse slide under flowing (tap water) • Dry slide with tissue from the edge then air-dry sample

Culture Recommendation • Thaw QUANTUM PBL (U11-022) and make aliquots (sterile tubes) • Thaw the pre-calculated amount of QUANTUM PBL medium (in tubes) until room temperature is reached • Transfer 0.5 ml of heparinised whole blood into the tube • Mix and incubate at +37 °C, 5% CO2 in an incubator for 48 to 72 hours • 1 – 2 hours before the end of the incubation period, add 0.1 ml of Colcemid (J01-003) (at a final concentration of 0.1 µg/ml) and mix gently • Preheat 0.075 M potassium chloride (S11-018) to 37 °C until used • Centrifuge (5 minutes at 500x g) • Discard the supernatant (leave a few drops at the bottom) • Add 5 – 10 ml of preheated potassium chloride (mix thoroughly while dispensing) • Leave for 10 minutes at room temperature • Centrifuge (5 minutes at 500x g) • Prepare fixative (freshly prepared 3 parts Methanol: 1 part Acetic acid) • Discard supernatant (leave a few drops at the bottom) • Add 5 – 8 ml of fixative • Repeat the last two steps (incubation and centrifugation) twice • Re-suspend the cell pellet in a small volume of fresh fixative

Application to Slide • • • • • Dry prepared slide on the downside Apply 3 drops of cell suspension and puff over the slide Draw slide 3 times through the yellow flame of a Bunsen burner Check through microscope If cells are too dense add a drop of fixative or use only two drops of cell suspension • Label slide

For in vitro laboratory use or further manufacturing only. Not for human use.

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