Pre-­equilibrate gel
1. Add gel to insert using positive displacement pipette
2. Add 750uL media on top of gel and in external well
3. Incubate for 20 minutes at 37 °C, 5 % CO2, humidified incubator
4. Remove media from on top of gel carefully
5. Remove media surrounding insert
6. Repeat steps 2 – 5 twice (3 media changes over 1 hour)
7. Alternatively, leave gel in media in the incubator between 1 hour and overnight
8. Prepare cells (cell concentration 0.5-­‐2 million cells/mL)
9. Remove media from gels
10. Pipette cells slowly onto the gel
11. Replace media around and on top of gel
12. Culture as usual

Seed cells on top of gel
1. Prepare cells (cell concentration 0.5-­2million cells/mL)
2. Aliquot maximum 2mL gel into an eppendorf, straight-­‐sided tubes are better than 1.5 mL pointy ones
3. Gently and slowly transfer 100uL cells onto the gel
4. Repeat with remaining 100uL
5. Add 750uL of media on top of gel and in external well
6. Incubate for 20 minutes at 37 °C, 5 % CO2, humidified incubator
7. Remove media from on top of gel carefully
8. Remove media surrounding insert
9. Repeat steps 4 – 7 twice (3 media changes over 1 hour)
10. Culture as usual

Table 1: Recommended volumes of PeptiGel for cell culture inserts or multi-­well plates

PeptiGel Volume

Number of wells    For insert (µL)    For plates (µL)
96                                  N/A                             35
24                                  150                             300
12                                  250                             400-­500