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Middle Aged (35-54)Accents
North American (General)Transcript
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how to concentrate and recover cells with the Give co cts roti a counter flow centrifuge ation system now that your cells are concentrated within the chamber, you are ready to recover them to an output bag. The primary objective of concentrate recovery is to recover all cells contained in the fluid. Ized bed depending on the application objectives might include minimizing wash buffer carryover, maximizing cell concentration or achieving a specific cell concentration or volume concentrate recovery is achieved by reversing the pump with the counter flow centrifuge, ation or CFC chamber still spinning the fluid. Ized bed is pulled from the tip of the chamber as a concentrated trail of cells in the medium that was used in the previous step lines, E. F and H. Are the preferred output lines, since cells can be delivered to an output bag without passing through the bubble trap. The following tips described the tools available for programming and optimizing the concentrate recovery sequence. First insert of 5-10 2nd, pause step prior to the recovery step to stabilize the fluid. Ized bed. If a smaller, more concentrated output volume is required, use this pause step to further concentrate the fluid Ized bed by increasing the G force or reducing the flow rate To ensure that the fluid eyes bed remains stable during the speed transition, insert a ramp time of 2-5 seconds. Your concentrated cells are now ready for recovery. The simplest method for concentrate recovery is to program a normal step where the pump is reversed and delivers a target volume to an output bag. Using wash buffer as the source fluid. Select a trigger volume and the target volume you wish to recover, which can be a fixed value or volume from the volume register. Use the same G. Force and a flow rate greater than or equal to the rate that was used in the pause step. The target volume recovered using this method includes not only the concentrate but also the dead volume in the tube between the tip of the cone and the output valve. The cts roti A system also includes a harvest step that enables you to minimize the dead volume in the fluid path between the tip of the CFC chamber and the output valve. Harvest step can only be selected if one of the output valves, E. F or H. Is active for the step and the pump is set in the reverse flow direction. The harvest step keeps the output valve closed until the cone to val volume has bypassed the valve. The default cone to Val volume is 0.5 ml and can be adjusted by the user to further optimize the harvest step. Since the harvest step delays opening of the output valve, the flow path of the pause step prior to the harvest step will be used to divert the dead volume. Once the cone to valve volume has been diverted, the output valve opens and the target volume will be delivered into the output bag. A typical harvest step. Using output valve H would proceed in this sequence, the pump direction will reverse and the cone to valve volume will be diverted past valve H. Exiting the T fitting to the left while the concentrated cells make their way toward the output valve. Once the pump has finished diverting the cone to valve volume, valve H will open and the concentrate will be delivered to the output bag after the desired volume is pumped into the output bag valve H will close and complete the harvest step. Whenever the harvest function is being used, an optical density sensor in the instrument measures the transmissibility of the media as it exits the CFC chamber. The start of concentrate and end of concentrate settings are used to automatically calculate the optical density sensor volume or O. D. Sensor volume which is available for selection in the volume. Register using the harvest function and the O. D. Sensor volume. Enable the cts roti a system to recover concentrated cells without dead volume or needing to know the volume of cells in the CFC chamber. If concentrate recovery is the last step in your protocol, add a subsequent 12th pause step with a flow rate of 10 ml per minute G. Force of 200 G and a five second ramp time to allow the instrument to smoothly ramp to a stop upon protocol completion. Watch the following video for tips and tools around process integrity C. T. A. For more information on the cts roti a counter flow centrifuge ation system visit thermo fisher dot com slash row tia.