PRODUCT: CyGEL Sustain™ PRESENTATION: aqueous solution. PRODUCT NUMBERS: CS20500 STORAGE: store at 2-8 2C. Do not freeze. CyGEL Sustain™ KEY CHARACTERISTICS: • Liquid below 20/21°C and gel above • Infinitely thermo-reversible • Refractive index: —1.37 • Optically clear and inert, without visible range auto-fluorescence • Viscosity increases with temp., between 21 and 28°C and plateaus beyond 37°C • Excipients modify the viscosity and the sohgel transition temperature • Recommended max. dilution: <20% v/v Table 1. Preparing RPMI culture medium for dedicated use with CyGEL™ Sustain To 20 ml 10X RPMI (Sigma:R1145), add:-• 2.0 ml PenicillinflO1 2 3 4 5 6 U) / Streptomycin (lOmg/ml) in 0.85% NaCI (lnvitrogen:15140-148) • 2.0 ml L-Glutamine 200 mM; (Sigma:G7513) • 5.4 ml 7.5% sodium bicarbonate soln. (Sigma:S8761) • 0.4 ml IN sodium hydroxide For a final volume of RPMI-stock of 29.8 ml DESCRIPTION: CyGEL Sustain™ is a novel thermo-reversible mountant that is compatible with extended imaging of live cells, tissues and organisms. It immobilizes non-adherent objects by simple warming and allowing their recovery by simple cooling. CyGEL Sustain™ is unusual in that it is a liquid when cold and a gel when warmed. CyGEL Sustain™ has ideal optical properties - low auto-fluorescence, R.l. -1.37, clear, and non-quenching. CyGEL Sustain™ is specially formulated to be culture-medium ready for extended imaging applications: 4 to 6 hours, depending on cell type. APPLICATIONS: • advanced microscopy / CLEM: extended imaging live de-adhered / non-adherent cells (Upton, 2007) imaging live tissues and spheroids (Robertson, 2010) imaging and manipulating live zebrafish embryos (Alvarez, 2009) BEFORE STARTING: Read the MSDS. Wear protective clothing, safety goggles and laboratory gloves. MATERIALS OFTEN REQUIRED BUT NOT SUPPLIED: Culture medium (e.g. 10X RPMI, antibiotics, glutamine, sodium bicarbonate, sodium hydroxide), plasticware, ice-bath/-pack, DRAQ5™, DRAQ7™, propidium iodide. EXAMPLE PROTOCOLS PROTOCOL 1: PREPARATION OF CyGEL Sustain™ FOR USE WITH RPMI 1640 As supplied, CyGEL Sustain™ will transit from sol to gel at 23-24°C. 1. Cool an unopened vial of CyGEL Sustain™ on ice for 1-2 minutes. 2. Using a sterile pipette tip, add 87.5 pi of RPMI culture medium (see table 1). Mix thoroughly, taking care to avoid bubble formation. RPMI-primed CyGEL Sustain™ is now isotonically correct for addition of viable cells. It will now transit from sol to gel at 20-21°C, usually within 1-2 minutes. NOTE: The total volume of cells, beads and dyes added to the RPMI-primed CyGEL Sustain™ should not exceed 20% v/v. Over-dilution will result in loss of ability to gel. PROTOCOL 2: CyGEL Sustain™ MOUNTING OF CELLS ONTO A STANDARD MICROSCOPE SLIDE ©Copyright BioStatus 2014-2024. All Rights Reserved Cool an unused vial of RPMI-primed CyGEL Sustain™ on ice. Prepare cells for mounting in CyGEL Sustain™: Wash the cells in buffer (e.g. PBS) by centrifugation. Resuspend the cell pellet in a maximum of 50 pi buffer (1 - 5 x 105 cells depending upon the application). Pipette the cell suspension into the vial containing PBS-primed CyGEL Sustain™. Transfer 250ul of CyGEL Sustain™/cell suspension into a cold P1000 pipette tip. Quickly dispense onto a clean slide by streaking along the surface for the length of coverslip to be applied (see fig. 1). Repeat the process for the remaining CyGEL Sustain™/cell suspension - giving two microscope slide preparations. Apply a coverslip to each CyGEL Sustain™/cell suspension "button" (see fig. 2). Place microscope slides onto an ice pack to allow the CyGEL Sustain™ to liquefy whereupon the sample will spread out under the coverslip. Remove the slide from the ice pack. The CyGEL Sustain™ will now re-set as it reaches room te

CyGEL Sustain™ TECHNICAL DATA SHEET PROTOCOL 3: CyGEL Sustain™ AS A DELIVERY MEDIUM FOR A CELL-PERMEANT DYE (DRAQ5™) IN FLUORESCENT IMAGING OF ADHERENT CELLS 1. Cool an unused vial of RPMI-primed CyGEL Sustain™. 2. Pipette 2.4 |il DRAQ5™ (5 mM stock) and dispense into the CyGEL Sustain™ and mix thoroughly. DRAQ5™ is now at a concentration of 20 |iM, sufficient for stoichiometric chromatin binding. 3. For the addition of cells, continue by following Protocol 2 above. DRAQ5™ nuclear staining should completely equilibrate after 60-80 min. However, sufficient staining should allow imaging of nuclei...