Secretome Collection

This protocol demonstrates how to collect secretome from the adipose tissue derived mesenchymal stem cells (ADMSCs). 

1. ADMSCs cultivation (Primarily cultured from adipose tissue) 
1.1 The cell stock was re-suspended with DMEM (Invitrogen, Life Technology, HK) with 10% FBS (Gibco, HK), 1% penicillin- streptomycin (Gibco, HK). 
1.2 Trypan blue was used to check cell viability under the microscope.
1.3 The cells were seeded in a 75cm² culture flask (Thermo Fisher Scientific, HK) for further experiment.

2. Secretome collection
2.1 When the cells were 90% confluent, they were washed with PBS for twice to remove FBS.
2.2 DMEM, without FBS, was added to the cells and incubated at 37°C with a humidified atmosphere of 5% CO₂ incubator for 18 hours. 
2.3 After 18 hours, the serum free medium was collected into 50 ml centrifuge tube and centrifuged at 1500 rpm for 10 minutes to remove debris.
2.4 Then the cell supernatant was transferred into centrifugal filter units with a 3 kDa molecular mass cut-off (Amicon® Ultra-15 Centrifugal Filter Units, Millipore, HK) and concentrated.
2.5 The filter units were centrifuged for 45 mins at the speed of 4000 rcf.
2.6 The concentrated solution was aliquoted into 1.5ml tubes (Eppendorf, HK) and stored in -80°C for further experiments. 

3. Materials

Dulbecco’s modified Eagle’s medium (DMEM)

Fetal bovine serum (FBS)

Penicillin- streptomycin

Phosphate buffered saline (PBS)

Centrifuge tubes

Amicon® Ultra-15 Centrifugal Filter Units