Posted on Jul 14, 2018

A 2D imaging experiment setup is preferred for un-labeled cell tracking of smaller cells such as immune cells. In MetaVi Labs' pathotaxis experiments when immune cells are co-cultured with pathogenic cells, 2D allows improved tracking of the immune cells (when the immune cells are un-labeled).

While tumor cells will adhere to glass or plastic, immune cells require a protein coating. Fortunately, this is easily accomplished with slides from ibidi.

Modifications to ibidi Protocols

We recommend ibidi slides and their associated protocols. However, please note:

  1. The ibidi recommendations for cell density are not applicable to many Pathotaxis experiments. We recommend finding the minimum cell densities possible to allow for greater accuracy in tracking and analysis, while still accomplishing the biological aim.
  2. For T-cell experiments, we recommend imaging at least one image per 30 seconds (or faster) if it is desired to track T-cell movement.
  3. In 2D experiments, cell adhesion should begin within 30 minutes of application of the cell media to the slide. Imaging should start at the 30-minute mark.

µ-Slide Chemotaxis

Used to investigate chemotaxis of fast or slow migrating adherent cells and non-adherent cells in gel matrices.

T-Cell Adhesion

This chamber can be used for non-traditional-chemotaxis experiments (i.e., experiments in which cell migration is important but an external chemo-attractant is not being used but rather the T-cells will be demonstrating Pathotaxis due to the pathogenic cells). This chamber has the advantage of a surface coating to which T-Cells will adhere. The product has two surface coating options, ibid-Treat, and Collagen IV. Other labs have had successful T-Cell adhesion with the Collagen IV surface coating.

Ordering Information

Catalog Number 80322

Surface Modification: Collagen IV: #1.5 polymer coverslip, sterilized.

Protocol

The ibidi Application Note 17 has a detailed protocol for 2D experiments.

Application Note 17: 2D and 3D Chemotaxis Assays Using µ-Slide Chemotaxis

µ-Slide 8-Well

An open µ-Slide (chambered coverslip) with 8 wells for use in immunofluorescence and high-end microscopy.

T-Cell Adhesion

This chamber has the advantage of a variety of surface coatings. Other labs have had successful T-Cell adhesion with the Collagen IV surface coating.

Ordering Information

Catalog Number 80822

Surface Modification: Collagen IV: #1.5 polymer coverslip, sterilized.

Protocol

The following link provides the detailed usage instructions for this versatile plate:

µ-Slide 8 Well Detailed Protocol

Custom Surface Coatings

The ibidi slides can also be ordered with either no-coating or the ibidi-Treat coating. This allows for creating your own coating.

Coating procedures for ibidi µ-Slides and µ-Dishes

For optimized cell adhesion, there are different treatments and coatings for the ibidi labware family. The ibiTreat surface is comparable to standard tissue culture treated labware. This surface permits direct cell growth with a large number of cell lines and primary cells. Compared to the ibiTreat surface, the uncoated surface is very hydrophobic. The uncoated surface must be coated with a protein solution for the adhesion of most cells.

Recommended Surfaces

  • For Collagen I: ibiTreat (tissue culture treated)
  • For Collagen IV: ibiTreat (tissue culture treated) or hydrophobic, uncoated
  • For Fibronectin: ibiTreat (tissue culture treated) or hydrophobic, uncoated
  • For Poly-L-Lysine: ibiTreat (tissue culture treated)
  • For Poly-D-Lysine: ibiTreat (tissue culture treated)

To establish a specific coating relevant to a specific research application, we recommend testing the coating procedure on both uncoated and ibiTreat µ-Slides. We have observed that some biomolecules adhere differently to hydrophobic and hydrophilic plastic surfaces. Please note that there is no uncoated version of the µ-Slide Chemotaxis. Use the ibiTreat surface for all coatings. Some products are also offered with a glass bottom. Glass can also be coated according to the specifications below. The ESS surface needs higher protein concentrations for an effective coating and must not be dried after coating.

Complete Application Note for Custom Surfaces

Reference Paper for T-cell Migration on 2D Surface

This open access paper describes T-cell migration mechanisms and utilized the ibidi µ-Slide Chemotaxis with Collagen IV surface treatment.

T Lymphocyte Migration: An Action Movie Starring the Actin and Associated Actors

A 2D imaging experiment setup is preferred for un-labeled cell tracking of smaller cells such as immune cells. In MetaVi Labs' pathotaxis experiments when immune cells are co-cultured with pathogenic cells, 2D allows improved tracking of the immune cells (when the immune cells are un-labeled).

While tumor cells will adhere to glass or plastic, immune cells require a protein coating. Fortunately, this is easily accomplished with slides from ibidi.

Modifications to ibidi Protocols

We recommend ibidi slides and their associated protocols. However, please note:

  1. The ibidi recommendations for cell density are not applicable to many Pathotaxis experiments. We recommend finding the minimum cell densities possible to allow for greater accuracy in tracking and analysis, while still accomplishing the biological aim.
  2. For T-cell experiments, we recommend imaging at least one image per 30 seconds (or faster) if it is desired to track T-cell movement.
  3. In 2D experiments, cell adhesion should begin within 30 minutes of application of the cell media to the slide. Imaging should start at the 30-minute mark.

µ-Slide Chemotaxis

Used to investigate chemotaxis of fast or slow migrating adherent cells and non-adherent cells in gel matrices.

T-Cell Adhesion

This chamber can be used for non-traditional-chemotaxis experiments (i.e., experiments in which cell migration is important but an external chemo-attractant is not being used but rather the T-cells will be demonstrating Pathotaxis due to the pathogenic cells). This chamber has the advantage of a surface coating to which T-Cells will adhere. The product has two surface coating options, ibid-Treat, and Collagen IV. Other labs have had successful T-Cell adhesion with the Collagen IV surface coating.

Ordering Information

Catalog Number 80322

Surface Modification: Collagen IV: #1.5 polymer coverslip, sterilized.

Protocol

The ibidi Application Note 17 has a detailed protocol for 2D experiments.

Application Note 17: 2D and 3D Chemotaxis Assays Using µ-Slide Chemotaxis

µ-Slide 8-Well

An open µ-Slide (chambered coverslip) with 8 wells for use in immunofluorescence and high-end microscopy.

T-Cell Adhesion

This chamber has the advantage of a variety of surface coatings. Other labs have had successful T-Cell adhesion with the Collagen IV surface coating.

Ordering Information

Catalog Number 80822

Surface Modification: Collagen IV: #1.5 polymer coverslip, sterilized.

Protocol

The following link provides the detailed usage instructions for this versatile plate:

µ-Slide 8 Well Detailed Protocol

Custom Surface Coatings

The ibidi slides can also be ordered with either no-coating or the ibidi-Treat coating. This allows for creating your own coating.

Coating procedures for ibidi µ-Slides and µ-Dishes

For optimized cell adhesion, there are different treatments and coatings for the ibidi labware family. The ibiTreat surface is comparable to standard tissue culture treated labware. This surface permits direct cell growth with a large number of cell lines and primary cells. Compared to the ibiTreat surface, the uncoated surface is very hydrophobic. The uncoated surface must be coated with a protein solution for the adhesion of most cells.

Recommended Surfaces

  • For Collagen I: ibiTreat (tissue culture treated)
  • For Collagen IV: ibiTreat (tissue culture treated) or hydrophobic, uncoated
  • For Fibronectin: ibiTreat (tissue culture treated) or hydrophobic, uncoated
  • For Poly-L-Lysine: ibiTreat (tissue culture treated)
  • For Poly-D-Lysine: ibiTreat (tissue culture treated)

To establish a specific coating relevant to a specific research application, we recommend testing the coating procedure on both uncoated and ibiTreat µ-Slides. We have observed that some biomolecules adhere differently to hydrophobic and hydrophilic plastic surfaces. Please note that there is no uncoated version of the µ-Slide Chemotaxis. Use the ibiTreat surface for all coatings. Some products are also offered with a glass bottom. Glass can also be coated according to the specifications below. The ESS surface needs higher protein concentrations for an effective coating and must not be dried after coating.

Complete Application Note for Custom Surfaces

Reference Paper for T-cell Migration on 2D Surface

This open access paper describes T-cell migration mechanisms and utilized the ibidi µ-Slide Chemotaxis with Collagen IV surface treatment.

T Lymphocyte Migration: An Action Movie Starring the Actin and Associated Actors

Posted on May 7, 2018

Un-labeled Cell Tracking Demo labeled image

Un-labeled Cell Tracking Demo labeled

Motion Stabilization

Before (un-stabilized):

After (stabilized):

Posted on Apr 15, 2018

We have made good progress thanks to our alpha-test partners supplying excellent samples. We have algorithms that now track immune cells and pathogen cells when each is captured on an independent image channel (i.e. two labels).

We are also extracting mini-movies of each pathogen around the time of an attack. We indicate the attack with a yellow circle. The yellow cross-hair indicates the center of the pathogen cell. Samples and a full report link are below.

We next will start working on fine-tuning the parameters that define an attack event such as how long it must last, better resolution of contact between the two cells, etc.

Full report here. Each report will contain the pathogen cell track videos (samples below). These can be found in the Well Details tab.

For best viewing click on the full-screen icon in each movie. The radar plots associated with each pathogen track movie are clickable to get full-screen versions.

pathogen cell 11 plot chart

pathogen cell 13 plot chart

pathogen cell 16 plot chart

pathogen cell 22 plot chart

Below is the full track movie and the original, un-processed movie.

Posted on Mar 29, 2018

Posted on Mar 29, 2018

Posted on Feb 5, 2018

Below are a few sample chemotaxis movies that were created from real lab well images. This should give you an idea of how we can help your lab and research efforts. Contact us if you have any questions at all, or would like to get a free demonstration using your own well images.

Fibroblasts in an ibidi µ-Slide chamber.

MDA-MB-231 in 3D Collagen, Treated with Propranolol.

MDA-MB-231 in 3D Collagen, Control (no treatment).

Neutrophils on 2D, Bright-field

Neutrophils with Aspergillus.

Posted on Dec 7, 2017

Abstract

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL), the functional coordination of which are governed by various signal substances, are crucial in the body’s defense of tumor and virus-infected cells. We investigated the role of various neurotransmitters and hormones on the regulation of functional parameters, including NK cell cytotoxicity, and the migration of NK cells and CTL within a three-dimensional collagen lattice. Using peripheral blood CTL and NK cells, we show that the neurotransmitters endorphin, histamine and substance P increase NK cell cytotoxicity, while norepinephrine inhibits cytotoxicity. Moreover, substance P reduces migratory activity, while norepinephrine increases NK cell and CTL migration. Furthermore, all three steroid hormones which were investigated, namely cortisone, testosterone, and estradiol, had regulatory influence on both cytotoxicity and migration of NK cells. These results further specify the functional basis of the complex interconnection between the immune and neuro-endocrine systems.

© 2003 Elsevier B.V. All rights reserved.

Keywords: Neurotransmitters; Natural killer cells; Cytotoxic T lymphocytes; Cell migration; Cytotoxicity; Activation; Interleukin-2

Abstract

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL), the functional coordination of which are governed by various signal substances, are crucial in the body’s defense of tumor and virus-infected cells. We investigated the role of various neurotransmitters and hormones on the regulation of functional parameters, including NK cell cytotoxicity, and the migration of NK cells and CTL within a three-dimensional collagen lattice. Using peripheral blood CTL and NK cells, we show that the neurotransmitters endorphin, histamine and substance P increase NK cell cytotoxicity, while norepinephrine inhibits cytotoxicity. Moreover, substance P reduces migratory activity, while norepinephrine increases NK cell and CTL migration. Furthermore, all three steroid hormones which were investigated, namely cortisone, testosterone, and estradiol, had regulatory influence on both cytotoxicity and migration of NK cells. These results further specify the functional basis of the complex interconnection between the immune and neuro-endocrine systems.

© 2003 Elsevier B.V. All rights reserved.

Keywords: Neurotransmitters; Natural killer cells; Cytotoxic T lymphocytes; Cell migration; Cytotoxicity; Activation; Interleukin-2