By Cheryl D. Helgason (auth.), Cheryl D. Helgason, Cindy L. Miller (eds.)
In this totally revised version of a longtime vintage, specialist researchers and clinicians describe in step by step element up to date ideas for the isolation and progress of the foremost fundamental telephone kinds, resembling kidney proximal tubule cells, hepatocytes, keratinocytes, and cardiomyocytes. The authors supply easily reproducible new tools for the differentiation of embryonic stem (ES) cells into numerous hematopoietic phone varieties, for fetal thymic organ tradition, and for the isolation and tradition of specialised cellphone varieties, corresponding to mammary progenitor cells, skeletal muscle myofibers, mesenchymal cells, neural stem cells, hematopoietic cells, stromal telephone traces, and endothelial cells. extra chapters describe new recommendations (leukocyte rolling, isolation of facet inhabitants cells, and scalable construction of ES-derived cells) and aspect quality controls tools for cellphone strains (detection and removal of mycoplasma, DNA fingerprinting, and cytogenetic analysis). The protocols stick with the winning tools in Molecular Biology™ sequence layout, each one delivering step by step laboratory directions, an creation outlining the primary in the back of the strategy, lists of the required gear and reagents, and tips about troubleshooting and keeping off recognized pitfalls.
updated and hugely functional, simple phone tradition Protocols, 3rd version, deals simple scientists and clinician-researchers strong instruments to isolate, tradition, and signify the promising really good mobile forms popular today.
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Additional info for Basic Cell Culture Protocols
Per reaction, 8 ilL of the amplified DNA is directly taken from the PCR reaction and is digested in parallel reactions with the restriction endonucleases AspI, HaeIII, HpaII, and XbaI by the addition of 1 ilL of the appropriate lOX restriction enzyme buffer and 1 ilL of the restriction enzyme. The mycoplasma species can be determined directly by the restriction pattern (see Fig. 2). This analysis allows only the determination of those mycoplasma species that most often (>98%) occur in cell cultures and is not suitable for the global identification of all types of mycoplasma species.
Discard the prehybridization solution and replace it with 5-10 mL of prewarmed hybridization solution containing 10-20 pmoUmL of digoxigenin-labeled oligonucleotide GTGs (see Note 9). 4. After incubation overnight with rotating, the hybridization solution is discarded and replaced with prewarmed wash buffer I; wash the membrane for 25 min at 42°C and repeat this step. 5. Next, wash the membrane twice (10 min each time) using prewarmed wash buffer II. Proceed to the detection procedure or store the membrane in wash buffer I at 4°C.
7. , Uphoff, C. , and Drexler, H. G. (1993) Specificity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines. J. ImmunoI. Methods 164,91-100. 3 _ Eradication of Mycoplasma Contaminations Cord C. Uphoff and Hans G. Drexler Summary Mycoplasma contaminations have a multitude of effects on cultured cell lines that can potentially influence the results of experiments and pollute bioactive substances used in human medicine.
Basic Cell Culture Protocols by Cheryl D. Helgason (auth.), Cheryl D. Helgason, Cindy L. Miller (eds.)