Overview¶
Embedding: Thermal Setting holds a sample in an agarose gel that sets on cooling. The agarose is dissolved hot in the outer solution and kept warm until the sample is mixed in. This page covers Gel: ULGA and Gel: LGA, which share the steps and differ in the parameters below.
Schematic representation of unilamellar liposomes held in a hydrogel matrix. Figure from Understanding Liposome Behaviour in Hydrogel Matrices.
Parameters of each gel.
| Parameter | Gel: ULGA | Gel: LGA |
|---|---|---|
| Stock concentration | 10 mg/mL (1.0 % w/v) | 28 mg/mL (2.8 % w/v) |
| Dissolve | Microwave | 95 °C heat block |
| Hold until use | 55 °C bead bath | 45 °C dry bath |
| Stock per well, before mixing | 15 µL of stock | 7.5 µL of stock and 7.5 µL of outer solution |
| Final concentration | 5 mg/mL (0.5 % w/v) | 7 mg/mL (0.7 % w/v) |
| Gel point | About 8 °C to 15 °C | About 24 °C to 28 °C |
Materials and Equipment¶
The agarose is listed under Materials on Gel: ULGA and Gel: LGA.
Reagents.
Equipment.
| Item | Used for |
|---|---|
| Microwave | Dissolving ULGA |
| 95 °C heat block | Dissolving LGA |
| 55 °C bead bath | Holding ULGA |
| Dry bath | Holding LGA at 45 °C |
| Vortex mixer | Dissolving LGA |
| 96-well or 384-well plate | Plating |
Protocol¶
Prepare the Outer Solution¶
Prepare the outer solution.
Use the outer solution the sample was prepared in. Its composition is flexible: match its osmolarity to the cells being embedded, rather than following one fixed recipe. The supporting DevNotes used glucose and 50 mM HEPES, at a final osmolarity of about 1200 mOsm, as their reference outer solution.
Prepare the ULGA Stock¶
Dissolve the ULGA.
Prepare ULGA at 10 mg/mL (1.0 % w/v) in the outer solution.
Microwave in 10 s bursts, 5–7 times, until the ULGA is fully dissolved.
Hold the stock.
Keep the stock on a 55 °C bead bath until use.
Prepare the LGA Stock¶
Dissolve the LGA.
Dissolve LGA at 28 mg/mL (2.8 % w/v) in the outer solution, adjusting to a final osmolarity of about 1200 mOsm.
Heat the solution in a 95 °C heat block until the LGA is fully dissolved. Vortex occasionally to help it dissolve.
Hold the stock.
Transfer an aliquot to a tube and keep it on a 45 °C dry bath until use.
LGA can set at room temperature
Keeping the stock warm prevents it from setting before it is mixed with the sample.
Dilute the stock for each well.
Combine 7.5 µL of the LGA stock with 7.5 µL of the outer solution.
Mix with the Sample and Plate¶
Mix the sample with the gel precursor.
For ULGA, combine 15 µL of sample with 15 µL of the ULGA stock.
For LGA, combine 15 µL of sample with the 15 µL of diluted LGA stock at 45 °C.
Plate the mixture.
Dispense 30 µL per well into a 96-well or 384-well plate. Plate each sample in duplicate.
Plate the sample in its outer solution, with no gel, as a reference.
Supported by¶
DevNotes that support this page include:
