Zeiss 510 multiphoton laser scanning microscope. To block N-cadherin, myeloma cells, 5T33MM and 5TGM1, were incubated with anti-N-cadherin 40 µg/ml antibody (Sigma, UK) for 30 minutes at 37 °C (). Mouse IgG1 antibody (Dako, Denmark) was used as a negative control. Cells were mixed every 5 minutes. After 30 minutes, myeloma cells were centrifuged at 1000 rpm for 4 minutes, washed twice with PBS to remove the antibody and re-suspended in 1 ml PBS. Cells were counting (see section 2.2.1) to determine the percentage of dead and live cells after blocking. Inoculation of MM cells into mice 4-6 week old male C57BL/KaLwRij mice were used. Mice were handled carefully. Mice were put in rodent restrainer, a plastic that hold mice body and keep their tails free for induction, and their tails were anesthetized using EMLATM cream 5% (AstraZeneca, UK). After 5 minutes, 200 µl of MM cell lines: 5T33MM and 5TGM1 (2x106 cells/200 µl) were inoculated into each mouse via their tail veins. Mice were harvested after various times according to the aims of each study.
Zeiss 510 multiphoton laser scanning microscope. To block N-cadherin, myeloma cells, 5T33MM and 5TGM1, were incubated with anti-N-cadherin 40 µg/ml antibody (Sigma, UK) for 30 minutes at 37 °C (). Mouse IgG1 antibody (Dako, Denmark) was used as a negative control. Cells were mixed every 5 minutes. After 30 minutes, myeloma cells were centrifuged at 1000 rpm for 4 minutes, washed twice with PBS to remove the antibody and re-suspended in 1 ml PBS. Cells were counting (see section 2.2.1) to determine the percentage of dead and live cells after blocking. Inoculation of MM cells into mice 4-6 week old male C57BL/KaLwRij mice were used. Mice were handled carefully. Mice were put in rodent restrainer, a plastic that hold mice body and keep their tails free for induction, and their tails were anesthetized using EMLATM cream 5% (AstraZeneca, UK). After 5 minutes, 200 µl of MM cell lines: 5T33MM and 5TGM1 (2x106 cells/200 µl) were inoculated into each mouse via their tail veins. Mice were harvested after various times according to the aims of each study.
PhD%20thesis%20%28Osama%20Al-Amer%29.docx