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CyGEL™ protocols

Example protocols for different imaging applications using CyGEL™

- the EASY way to visualise suspension cells ...

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THE FOLLOWING PROTOCOLS ARE AVAILABLE:

  • CyGEL™ MOUNTING OF CELLS ONTO A STANDARD MICROSCOPE SLIDE FOR CONFOCAL MICROSCOPY / CELL IMAGING
  • CyGEL™ AS A DELIVERY MEDIUM FOR A CELL-PERMEANT DYE (DRAQ5™) IN FLUORESCENT IMAGING OF ADHERENT CELLS
  • CyGEL™ AS A DELIVERY MEDIUM FOR A CELL-IMPERMEANT DYE (e.g. PROPIDIUM IODIDE) IN TIME-LAPSED FLUORESCENT IMAGING OF MEMBRANE-COMPROMISED CELLS
  • CyGEL™ MOUNTING OF CELLS IN A CHAMBER COVERSLIP FOR CONFOCAL MICROSCOPY / CELL IMAGING

and can be reviewed below .........


General tips:

Read the material safety data sheet supplied prior to use of CyGEL™

CyGEL™ changes from a sol (chilled) to a gel (warmed) quickly, aiding rapid sample preparation and immobilisation of cells for visualisation.

CyGEL™ becomes a gel at a convenient temperature (nominally room temperature).

CyGEL™ is formulated to maintain cellular integrity for periods of ≥ 1 h for most cell types and often much longer.

Keep CyGEL™ stocks on ice to facilitate pipetting.

Pre-chill tips / pipettes by storing at -20°C or draw and eject ice cold water through the tip immediately prior to use.

Ice cold water can be used to "clear" clogged / blocked tips.

P1000 micropipette displacement tips are recommended for handling CyGEL™.

CyGEL™ hydrogel can accept additives such as fluorescent DNA dyes (e.g. DRAQ5™, propidium iodide). These should be added when CyGEL™ is chilled (sol). Additions of 1:100 - 1:20 should not affect the performance of CyGEL™ but each new additive should be tested for impact on gel formation / stability. (See table of examples).

The cell suspension volume should not dilute CyGEL™ by more than 20%. Otherwise, the integrity of the hydrogel will be compromised.

CyGEL™ is intended for research purposes only.


PROTOCOL 1:

CyGEL™ MOUNTING OF CELLS ONTO A STANDARD MICROSCOPE SLIDE FOR CONFOCAL MICROSCOPY / CELL IMAGING

Reagents required:

  • CyGEL™
  • chilled water
  • chilled pipettetips
  • ice pack
  • microscope slides
  • coverslips
  • cell buffer e.g. PBS

Read the supplied Material Safety Data Sheet prior to handling CyGEL™.

  1. Cool a vial of CyGEL™ on ice / ice pack. Transfer 250 µl with a P1000 pipette tip to a clean microcentrifuge tube and place on ice.
  2. Prepare cells for mounting in CyGEL™: Wash the cells in buffer (e.g. PBS) by centrifugation. Resuspend the cell pellet in 10 µl buffer (typically 1 - 5 x 105 cells depending upon the application).
  3. Pipette the cell suspension into the microcentrifuge tube containing CyGEL™.
  4. Transfer the total CyGEL™/cell suspension volume (ca. 260 µl) into a cold P1000 pipette tip. Quickly dispense it onto a clean microscope slide by streaking it along the surface for a length approaching that of the coverslip to be applied.
  5. Apply a coverslip to the CyGEL™/cell suspension.
  6. Place the microscope slide onto an ice pack to allow the CyGEL™ to liquefy. The sample will then spread out under the coverslip.
  7. Remove the slide from the ice pack. The CyGEL™ will now re-set as it warms towards room temperature. The sample is now ready for visualisation.

PROTOCOL 2:

CyGEL™ AS A DELIVERY MEDIUM FOR A CELL-PERMEANT DYE (DRAQ5™) IN FLUORESCENT IMAGING OF ADHERENT CELLS

Reagents required:

  • CyGEL™
  • chilled water
  • chilled pipette tips
  • cell / nuclear dye e.g. DRAQ5™
  1. Read the supplied Material Safety Data Sheet prior to handling CyGEL™.
  2. Cool a 1 ml vial of CyGEL™ on ice / ice pack.
  3. Pipette 4 µl‡ DRAQ5™ into the vial of chilled CyGEL™ and mix thoroughly. DRAQ5™ is now at a concentration of 20 µM, sufficient for stoichiometric chromatin binding.
  4. Prepare cells for mounting in CyGEL™: remove the culture medium by aspiration.
  5. Overlayer the cells with 250 µl of CyGEL™ / DRAQ5™ (for an 8-chamber coverslip).
  6. Allow the sample to warm up and image at room temperature.

‡ Adjust quantity of DRAQ5 according to the volume of CyGEL to be used

The individual nuclear fluorescence intensity with DRAQ5™ for each cell measured will reflect the cell cycle age distribution across the population.

PROTOCOL 3:

CyGEL™ AS A DELIVERY MEDIUM FOR A CELL-IMPERMEANT DYE (e.g. PROPIDIUM IODIDE) IN TIME-LAPSED FLUORESCENT IMAGING OF MEMBRANE-COMPROMISED CELLS

Reagents required:

  • CyGEL™
  • chilled water
  • chilled pipette tips
  • cell impermeant dye e.g. propidium iodide (PI), PBS
  1. Read the supplied Material Safety Data Sheet prior to handling CyGEL™.
  2. Cool a vial of CyGEL™ on ice / ice pack. Transfer 250 µl with a P1000 pipette tip to a clean microcentrifuge tube and place on ice.
  3. Prepare a stock solution of PI at 50 µg / ml
  4. Pipette 5 µl of 50 µg / ml PI into the tube of chilled CyGEL™ and mix thoroughly. PI is now at a concentration of 1 µM.
  5. Prepare cells for mounting in CyGEL™ / PI: Wash the cells in buffer (e.g. PBS) by centrifugation. Resuspend the cell pellet in 10 µl buffer (typically 1 - 5 x 105 cells depending upon the application).
  6. Pipette the cell suspension into the microcentrifuge tube containing CyGEL™ / PI.
  7. Transfer the total CyGEL™ / PI / cell suspension volume (ca. 260 µl) into a cold P1000 pipette tip. Quickly dispense it onto a clean microscope slide by streaking it along the surface for a length approaching that of the coverslip to be applied.
  8. Apply a coverslip to the CyGEL™ / PI / cell suspension.
  9. Place the microscope slide onto an ice pack to allow the CyGEL™ to liquefy. The sample will then spread out under the coverslip.
  10. Remove the slide from the ice pack. The CyGELTM will now re-set as it warms towards room temperature. The sample is now ready for visualisation.

Membrane-compromised (i.e. dying / apoptotic) cells will no longer be able to exclude PI and will appear fluorescent under excitation with the appropriate wavelength.

 

PROTOCOL 4:

CyGEL™ MOUNTING OF CELLS IN A CHAMBER COVERSLIP FOR CONFOCAL MICROSCOPY / CELL IMAGING

Reagents required:

  • CyGEL™
  • chilled water
  • chilled pipette tips
  • ice pack
  • microscope 8-chamber coverslip (e.g. Nunc)
  • cell buffer e.g. PBS
  1. Read the supplied Material Safety Data Sheet prior to handling CyGEL™.
  2. Cool a vial of CyGEL™ on ice / ice pack.
  3. Prepare cell suspension: Wash the cells in buffer (e.g. PBS) by centrifugation. Resuspend the cell pellet in the same buffer at a suggested concentration of 1 x 106 cells/ml.
  4. Pipette 50 µl of the cell suspension into a clean chamber of a microscope 8-chamber coverslip.
  5. Transfer 250 µl CyGEL™ with a P1000 pipette tip and directly overlayer the cells in the chamber.
  6. Allow the chamber to warm up to room temperature. The CyGEL™ layer will set thereby immobilizing cells and cell clusters for visualisation.

CyGEL™ is intended for research purposes only.