By George P. Studzinski
This article presents a special mix of succinctly expressed uncomplicated techniques of phone development and mobile demise with certain directions and protocols on how one can degree appropriately those methods. functional directions are observed by means of explanatory fabric which permits the researcher to decide on which specific protocol is healthier for his or her function. The tools defined variety from uncomplicated suggestions, resembling autoradiography and mobile staining, to extra advanced recommendations, comparable to movement cytometry.
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3. 6 uCi) to each well. Incubate at 37°C for 4-5 h. 4. After incubation, scrape the cells with a cell masher and aspirate along with the medium on to a Whatman filter. Wash the wells with distilled water extensively and aspirate through the Whatman filter to hypotonically lyse the cells and wash through the unincorporated tritiated thymidine. 5. Place each filter containing the bound DNA with incorporated thymidine from one well into a scintillation vial, add liquid scintillant, and count the rate of decomposition of tritium in a scintillation counter.
At the completion of the BrdU infusion, obtain a biopsy specimen of the tumour, fix in Bouin's solution for 3 h, dehydrate in ethanol and embed in plastic with glycol methacrylate, and cut 2 um sections using a microtome. 4. Rehydrate the slides with distilled water for 10 min. 5. Incubate the slides with 3% H2O2 for 30 min. 6. Incubate the slides with pronase 1 mg/mI for 45 min. 15 M PBS after each incubation. 7. 5 M PBS. 8. Sequentially stain slides with two monoclonal antibodies, one that recognizes BrdU only and one that recognizes both BrdU and lUdr.
G Robert Wieder Protocol 3. Continued Fixing cells 1. Centrifuge cells and aspirate off the PBS. 2. Add 2 ml 70% ethanol in water or PBS at 4°C for 1 h to fix cells. They may be stored for several days at 4°C at this point. 3. m. (600 x g) for 10 min, aspirate off the ethanol, and wash twice with 2 ml PBS. 4. Resuspend the cells in 1 ml PBS, add 10 ul RNase A (DNase-free), and incubate 37°C for 1 h. 5. m. (600 x g) for 10 min and aspirate supernatant. 6. Resuspend the cells in 500 ul of PI (50 ug/ml) in PBS and acquire the samples in the flow cytometer.