Download Multiple Myeloma: Methods and Protocols by Ross D. Brown, P. Joy Ho PDF

By Ross D. Brown, P. Joy Ho

A entire choice of vintage and cutting edge methodologies utilized in many laboratories for the research of a number of myeloma. those with no trouble reproducible thoughts variety from the normal Plasma mobilephone Labeling Index technique to a last bankruptcy on making feel of microarrays, and comprise the total spectrum of cytogenetic and molecular diagnostic equipment. The protocols stick with the winning equipment in Molecular Medicineв„ў sequence structure, each one supplying step by step laboratory directions, an advent outlining the primary at the back of the procedure, lists of the required gear and reagents, and pointers on troubleshooting and keeping off recognized pitfalls. those confirmed concepts are perfect for learning the pathogenesis of a number of myeloma and choosing new healing goals.

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2. Using a sterile pipet, inoculate the medium with an appropriate amount of bone marrow (see Note 2). 3. Lie the flask flat and incubate at 37°C in 5% CO2 for approx 72 h. 4. 2 mL (200 µL) of 10 µg/mL Colcemid to the culture and incubate at 37°C for a further 60 min. 5. 1. 2. Slide Making and G-Banding Slide preparation is important for optimal G-banding. Ideally, slides are made when the temperature is 22°C and the humidity is 40–45% (see Note 5). 1. Place the cell suspension on the bench and allow it to warm to room temperature.

Immunophenotyping of PCs in MM 23 6. The following commercially available reagent kits containing both permeabilization and fixation reagents can be applied as reference reagents: Fix & Perm (Caltag), Intraprep (Immunotech), Intrastain (DAKOCytomation). 7. For instrument setup follow the protocol proposed by EWGCCA guidelines (15). 8. In the case of samples corresponding to single cell suspensions that do not contain nonnucleated red cells, the lysing steps can be detected from protocols I (steps 5–8) and II (steps 5–8 of protocol I in step 8 of protocol II).

Because only a minority of metaphases from each culture may contain chromosome abnormalities, at least 20 metaphases from each culture should be fully analyzed. This may involve the scanning of more than one G-banded slide from each culture (see Note 14). 4. Notes 1. The G-banding method given here uses Leishman’s solution because we have found this method to be the most reliable and least given to fading over time. However, other methods commonly used in cytogenetics laboratories involve the use of Giemsa stain or Wright’s stain.

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