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Preparation of Metaphases - Engl

The document provides instructions for preparing metaphase chromosome spreads from blood samples for staining and microscopic analysis. The protocol involves culturing whole blood samples to stimulate cell division, treating with a chemical to arrest cells in metaphase, harvesting and fixing cells onto slides, and staining the slides with Giemsa stain to visualize individual chromosomes.

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35 views1 page

Preparation of Metaphases - Engl

The document provides instructions for preparing metaphase chromosome spreads from blood samples for staining and microscopic analysis. The protocol involves culturing whole blood samples to stimulate cell division, treating with a chemical to arrest cells in metaphase, harvesting and fixing cells onto slides, and staining the slides with Giemsa stain to visualize individual chromosomes.

Uploaded by

emmassr1198
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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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 g/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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