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Novel Cell and Tissue Acquisition System (CTAS): microdissection of live and frozen brain tissues.

Kudo LC, Vi N, Ma Z, Fields T, Avliyakulov NK, Haykinson MJ, Bragin A, Karsten SL - PLoS ONE (2012)

Bottom Line: We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics.High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting.We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

View Article: PubMed Central - PubMed

Affiliation: NeuroInDx, Inc., Signal Hill, California, United States of America. lckudo@neuroindx.com

ABSTRACT
We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

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Capillary-based vacuum-assisted cell and tissue acquisition system (CTAS) v.4.1.A. Representative image of CTAS v.4.1. The system is attached to an inverted microscope (TCM400, Labomed) and consists of the following key components: sample collection assembly with collection unit, LED lights, side chassis vacuum module and DCU controls. The latter incorporates electronic controls and a vacuum pump. The two dials at the front of the side chassis control the vacuum strength from 2″Hg (1) to maximum of 22″Hg (10) and the vacuum duration from 100 ms (1) to maximum of 1 second (10). Depending on the tissue type and section thickness, various vacuum strength and duration may be used. Green button turns the power on/off. Three DCU control buttons include two white buttons, which bring the DCU up or down during the calibration procedure and an orange button that sets the Home position of the DCU and brings it to its Standby position. Black “Sample” button initiates sample collection by bringing the DCU down to the Home position and activating the vacuum at the selected strength and duration; B. CTAS sample collection assembly in its lifted position for DCU attachment/removal. DCU attached to a collection unit with connectors for multiple cables and a vacuum tube. Calibration LED source for illuminating the tip of the capillary is shown. In this position, the green horizontal and red vertical calibration LED lights are automatically turned off. The lights are automatically turned on when the CTAS head is in its upright position. The x–y position of the DCU is controlled by the knobs on the linear stages (x–y controls).
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pone-0041564-g001: Capillary-based vacuum-assisted cell and tissue acquisition system (CTAS) v.4.1.A. Representative image of CTAS v.4.1. The system is attached to an inverted microscope (TCM400, Labomed) and consists of the following key components: sample collection assembly with collection unit, LED lights, side chassis vacuum module and DCU controls. The latter incorporates electronic controls and a vacuum pump. The two dials at the front of the side chassis control the vacuum strength from 2″Hg (1) to maximum of 22″Hg (10) and the vacuum duration from 100 ms (1) to maximum of 1 second (10). Depending on the tissue type and section thickness, various vacuum strength and duration may be used. Green button turns the power on/off. Three DCU control buttons include two white buttons, which bring the DCU up or down during the calibration procedure and an orange button that sets the Home position of the DCU and brings it to its Standby position. Black “Sample” button initiates sample collection by bringing the DCU down to the Home position and activating the vacuum at the selected strength and duration; B. CTAS sample collection assembly in its lifted position for DCU attachment/removal. DCU attached to a collection unit with connectors for multiple cables and a vacuum tube. Calibration LED source for illuminating the tip of the capillary is shown. In this position, the green horizontal and red vertical calibration LED lights are automatically turned off. The lights are automatically turned on when the CTAS head is in its upright position. The x–y position of the DCU is controlled by the knobs on the linear stages (x–y controls).

Mentions: CTAS v.4.1 is attached to an inverted TCM400 (Labomed) microscope and consists of the following major components: sample collection assembly, vacuum module and controls, two gooseneck LED lights and single axis stages (Fig. 1). A linear actuator is used for the vertical movement of the disposable capillary unit (DCU), providing precise positioning during calibration and sample acquisition (Fig. 1). DCU is connected to the vacuum line through the disposable filter unit (Millipore; Fig. 1). LED lights (Littlite) are used to illuminate the slide with a tissue sample (Fig. 1). Vacuum module incorporates electronic controls and a vacuum pump with pneumatic tubing (Fig. 1). Vacuum pump vibration mount ensures that linear actuator stability is not affected during pump operation. Two distinct vacuum pumps are used for the basic microdissection CTAS model v.4.1 (KNF Neuberger, MPU2568-NMP830-10.10) and CTAS-live tailored for isolation of live brain cells (KNF Neuberger, UN86KNDCB). Pump generating a stronger vacuum is required for the microdissection of generally thicker native tissues. Our current CTAS electrical circuit board is programmed to provide an appropriate range of the variable parameters for vacuum strength, duration and LED lights intensity. It incorporates the controls for the vertical movement of the linear actuator and positioning the tip of the DCU relative to the sample - calibration procedure (Fig. 1). The single-axis stages are used for positioning the DCU in the center of the microscope’s x-y stage (Fig. 1A–C). The outer casing of the CTAS components were designed in Solidworks and machined of aluminum alloy. The inverted microscope is equipped with a trinocular port for mounting a camera and/or video system with an appropriate adapter for process documentation or visualization via a computer monitor.


Novel Cell and Tissue Acquisition System (CTAS): microdissection of live and frozen brain tissues.

Kudo LC, Vi N, Ma Z, Fields T, Avliyakulov NK, Haykinson MJ, Bragin A, Karsten SL - PLoS ONE (2012)

Capillary-based vacuum-assisted cell and tissue acquisition system (CTAS) v.4.1.A. Representative image of CTAS v.4.1. The system is attached to an inverted microscope (TCM400, Labomed) and consists of the following key components: sample collection assembly with collection unit, LED lights, side chassis vacuum module and DCU controls. The latter incorporates electronic controls and a vacuum pump. The two dials at the front of the side chassis control the vacuum strength from 2″Hg (1) to maximum of 22″Hg (10) and the vacuum duration from 100 ms (1) to maximum of 1 second (10). Depending on the tissue type and section thickness, various vacuum strength and duration may be used. Green button turns the power on/off. Three DCU control buttons include two white buttons, which bring the DCU up or down during the calibration procedure and an orange button that sets the Home position of the DCU and brings it to its Standby position. Black “Sample” button initiates sample collection by bringing the DCU down to the Home position and activating the vacuum at the selected strength and duration; B. CTAS sample collection assembly in its lifted position for DCU attachment/removal. DCU attached to a collection unit with connectors for multiple cables and a vacuum tube. Calibration LED source for illuminating the tip of the capillary is shown. In this position, the green horizontal and red vertical calibration LED lights are automatically turned off. The lights are automatically turned on when the CTAS head is in its upright position. The x–y position of the DCU is controlled by the knobs on the linear stages (x–y controls).
© Copyright Policy
Related In: Results  -  Collection

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pone-0041564-g001: Capillary-based vacuum-assisted cell and tissue acquisition system (CTAS) v.4.1.A. Representative image of CTAS v.4.1. The system is attached to an inverted microscope (TCM400, Labomed) and consists of the following key components: sample collection assembly with collection unit, LED lights, side chassis vacuum module and DCU controls. The latter incorporates electronic controls and a vacuum pump. The two dials at the front of the side chassis control the vacuum strength from 2″Hg (1) to maximum of 22″Hg (10) and the vacuum duration from 100 ms (1) to maximum of 1 second (10). Depending on the tissue type and section thickness, various vacuum strength and duration may be used. Green button turns the power on/off. Three DCU control buttons include two white buttons, which bring the DCU up or down during the calibration procedure and an orange button that sets the Home position of the DCU and brings it to its Standby position. Black “Sample” button initiates sample collection by bringing the DCU down to the Home position and activating the vacuum at the selected strength and duration; B. CTAS sample collection assembly in its lifted position for DCU attachment/removal. DCU attached to a collection unit with connectors for multiple cables and a vacuum tube. Calibration LED source for illuminating the tip of the capillary is shown. In this position, the green horizontal and red vertical calibration LED lights are automatically turned off. The lights are automatically turned on when the CTAS head is in its upright position. The x–y position of the DCU is controlled by the knobs on the linear stages (x–y controls).
Mentions: CTAS v.4.1 is attached to an inverted TCM400 (Labomed) microscope and consists of the following major components: sample collection assembly, vacuum module and controls, two gooseneck LED lights and single axis stages (Fig. 1). A linear actuator is used for the vertical movement of the disposable capillary unit (DCU), providing precise positioning during calibration and sample acquisition (Fig. 1). DCU is connected to the vacuum line through the disposable filter unit (Millipore; Fig. 1). LED lights (Littlite) are used to illuminate the slide with a tissue sample (Fig. 1). Vacuum module incorporates electronic controls and a vacuum pump with pneumatic tubing (Fig. 1). Vacuum pump vibration mount ensures that linear actuator stability is not affected during pump operation. Two distinct vacuum pumps are used for the basic microdissection CTAS model v.4.1 (KNF Neuberger, MPU2568-NMP830-10.10) and CTAS-live tailored for isolation of live brain cells (KNF Neuberger, UN86KNDCB). Pump generating a stronger vacuum is required for the microdissection of generally thicker native tissues. Our current CTAS electrical circuit board is programmed to provide an appropriate range of the variable parameters for vacuum strength, duration and LED lights intensity. It incorporates the controls for the vertical movement of the linear actuator and positioning the tip of the DCU relative to the sample - calibration procedure (Fig. 1). The single-axis stages are used for positioning the DCU in the center of the microscope’s x-y stage (Fig. 1A–C). The outer casing of the CTAS components were designed in Solidworks and machined of aluminum alloy. The inverted microscope is equipped with a trinocular port for mounting a camera and/or video system with an appropriate adapter for process documentation or visualization via a computer monitor.

Bottom Line: We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics.High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting.We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

View Article: PubMed Central - PubMed

Affiliation: NeuroInDx, Inc., Signal Hill, California, United States of America. lckudo@neuroindx.com

ABSTRACT
We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

Show MeSH
Related in: MedlinePlus