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CyGEL Sustain™ protocols

Example protocols for different imaging applications using CyGEL Sustain

- the EASY way to visualise suspension cells for several hours in culture medium ...

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THE FOLLOWING PROTOCOLS ARE AVAILABLE:

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

- and are provided in this document.

- and are provided in this document.

General tips for use of CyGEL™ Sustain:

  • Read the material safety data sheet supplied prior to use of CyGEL™ Sustain.
  • CyGEL™ Sustain changes from a sol to a gel at 23-24°C, aiding rapid sample preparation and immobilisation of live cells for visualisation.
  • CyGEL™ Sustain is formulated to accept concentrated culture medium (e.g. RPMI) to maintain cellular integrity for periods of ≥ 2 h for most cell types and often much longer.
  • Keep CyGEL™ Sustain stocks refrigerated to facilitate convenient pipetting.
    If required, ice cold water can be used to "clear" clogged / blocked tips.  P1000 micropipette displacement tips are recommended for handling CyGEL™ Sustain.
  • CyGEL™ Sustain can accept additives such as fluorescent DNA dyes (e.g. DRAQ5™, propidium iodide). These should be added when CyGEL™ Sustain is chilled (sol). Additions of 1:100 - 1:20 should not affect the performance of CyGEL™ Sustain but each new additive should be tested for impact on gel formation / stability. (Examples can be found at www.biostatus.com ).  The overall cell/bead/dye suspension volume should not dilute CyGEL™ Sustain by more than 5%. Otherwise, the integrity of the hydrogel will be compromised.
  • CyGEL™ Sustain is intended for research purposes only. 

PROTOCOL 1: PREPARATION OF CyGEL™ Sustain FOR USE

Reagents required:
CyGEL™ Sustain
10X RPMI (See note a. below for formulation)
Microcentrifuge tubes
Pipette tips
ice pack/bath

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

As supplied, CyGEL™ Sustain will transit from sol to gel at approximately 23-24°C

  1. Remove a fresh vial of CyGEL™ Sustain.
  2. Chill the vial on ice.
  3. Add 87.5 μl RPMI-stock (as prepared in note a. below) to the CyGEL™ Sustain. Mix thoroughly, taking care to avoid bubble formation.

The RPMI-primed CyGEL™ Sustain is now at the correct isotonic strength for the addition of viable cells, bead and dyes up to a maximum volume of 5% v/v.
The RPMI-primed CyGEL™ Sustain will now transit from sol to gel at approximately 22-23°C.

Note a.
Preparation of RPMI culture medium for dedicated use with CyGEL™ Sustain
To 20 ml of 10X RPMI (Sigma:R1145), add:-
2.0 ml Penicillin(104 U)/Streptomycin(10mg/ml) in 0.85% NaCl (Invitrogen:15140-148)
2.0 ml L-Glutamine 200 mM; (Sigma:G7513)
5.4 ml 7.5% sodium bicarbonate soln. (Sigma:S8761)
0.4 ml 1N sodium hydroxide
For a final volume of RPMI-stock of 29.8 ml

PROTOCOL 2: CyGEL™ Sustain MOUNTING OF CELLS IN A CHAMBER COVERSLIP FOR CONFOCAL MICROSCOPY / CELL IMAGING

Reagents required:
RPMI-primed CyGEL™ Sustain (from Protocol 1)
microcentrifuge tubes
ice pack
microscope 8-chamber coverslip (e.g. Nunc)
cell buffer e.g. RPMI

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

  1. Cool a vial of RPMI-primed CyGEL™ Sustain to below room temperature.
  2. Prepare cell suspension: Wash the cells in buffer (e.g. RPMI) by centrifugation.
  3. Resuspend the cell pellet in the same buffer at a suggested concentration of 4 x 106 cells/ml.
  4. Pipette 12 μl of the cell suspension into a clean chamber of a microscope 8-chamber coverslip.
  5. Transfer 250 μl of the RPMI-primed CyGEL™ Sustain with a P1000 pipette tip and directly overlay the cells in the chamber.
  6. Warm the chamber above room temperature (e.g. on a thermally-controlled stage).

The CyGEL™ Sustain layer will set, thereby immobilizing cells and cell clusters for visualisation.

PROTOCOL 3: CyGEL™ Sustain AS A DELIVERY MEDIUM FOR A CELL-PERMEANT DYE (DRAQ5™) IN FLUORESCENT IMAGING OF ADHERENT CELLS

Reagents required:
RPMI-primed CyGEL™ Sustain (from Protocol 1)
chilled water
chilled pipette tips
Microcentrifuge tubes
cell / nuclear dye e.g. DRAQ5™

Read the supplied Material Safety Data Sheet prior to handling CyGEL™ Sustain and DRAQ5™.

  1. Cool a vial of RPMI-primed CyGEL™ Sustain below room temperature.
  2. Add 2.35 μl DRAQ5™ (5 mM stock) and mix thoroughly. DRAQ5™ is now at a concentration of 20 μM, sufficient for stoichiometric chromatin binding.
  3. For the addition of cells, continue by following Protocol 2 above.

DRAQ5™ nuclear binding should completely equilibrate after 60-80 min. However, imaging of nuclei will be possible after 20-30 min.

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

PROTOCOL 4: CyGEL™ Sustain AS A DELIVERY MEDIUM FOR A CELL-IMPERMEANT DYE (e.g. PROPIDIUM IODIDE) IN TIME-LAPSED FLUORESCENT IMAGING OF MEMBRANE-COMPROMISED CELLS

Reagents required:
RPMI-primed CyGEL™ Sustain
chilled water
chilled pipette tips
Microcentrifuge tubes
cell impermeant dye e.g. propidium iodide (PI), PBS

Read the supplied Material Safety Data Sheet prior to handling CyGEL™ Sustain and DRAQ5™.

  1. Cool the tube of RPMI-primed CyGEL™ Sustain
  2. Prepare a 1mg/ml stock solution of propidium iodide.
  3. Add 2.94 μl propidium iodide stock solution to the RPMI-primed CyGEL™ Sustain and mix thoroughly. Propidium iodide is now at a concentration of 5 μg/ml, sufficient for stoichiometric chromatin binding.
  4. For the addition of cells, continue by following Protocol 2 above.

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.

CyGEL™ Sustain is intended for research purposes only.