Adipose Tissue Dissociation Kit mouse and rat (Miltenyi Biotec; 130-105-808)

Kit Components: 5 vials containing:

2 vials of Enzyme D (lyophilized powder)

1 vial of Enzyme R (lyophilized powder)

1 vial of Enzyme A (lyophilized powder)

1 mL of Buffer A

Fibroblast Media (made ahead of time)

500mL MEMα (Gibco; 32561037)

10µL EGF 500 µL/mL (R&D Systems; 263-EG-200 = recon in 400 µL PBS, aliquot and store at -80C )

2.5 µL FGFbasic 50 µg/mL (R&D Systems; 233-FB-025 = recon in 500 µL PBS, aliquot and store at -80C)

50 mL FBS

5 mL MEM Non-essential amino acids (NEAA) (Gibco;11140050)

5 mL Pen/Strep (Gibco;15140122)

Prepare Enzyme D by reconstitution of the lyophilized powder in each vial with 3 mL of DMEM and sterile filter. Prepare 0.5 mL aliquots to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months after reconstitution.

Prepare Enzyme R by reconstitution of the lyophilized powder in the vial with 2.7 mL of DMEM. Prepare 250 µL aliquots to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months after reconstitution.

Prepare Enzyme A by reconstitution of the lyophilized powder in the vial with 1 mL of Buffer A supplied with the kit. Do not vortex. Prepare 50 µL aliquots to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months after reconstitution.

Prepare Enzyme Mix with prepared enzymes from step 1. 2-4 samples are typically processed at a

time and enzyme mix should be prepared as master mix for all the samples.

Enzyme Mix:

A	B	C
DMEM	2.35 mL
Enzyme D	100 µL
Enzyme R	50 µL
Enzyme A	12.5 µL
Total	2.5 mL

Aliquot 2.5 mL of Enzyme mix into gentleMACS C Tube for each sample and label tube.

Transfer 0.5g of adipose tissue into the gentleMACS C Tube containing the enzyme mix. Use a small sterile scissors to cut tissue into small pieces while in the C Tube. Tightly close tube beyond the first resistance.

Use the heating function of the gentleMACS Octo Dissociator with Heaters run program 37C_mr_ATDK_1 .

After termination of the program, detach C Tube from the gentleMACS Dissociator.

(Optional) Perform a short centrifugation step up to 300×g to collect the sample material at the tube bottom and resuspend cells.

Label a 15 mL tube for each sample. Place a MACS SmartStrainer (100 µm) on each 15 mL tube.

Pour the cell suspension through the MACS SmartStrainer (100 µm) into the labeled 15 mL tube.

Rinse the C tube and wash the MACS SmartStrainer with 5–10 mL of DMEM for each sample.

Discard the MACS SmartStrainer and centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.

Gently resuspend cell pellet with 1 mL ACK Lysis Solution (Gibco A10492-01) to lyse red blood cells. Incubate samples at room temperature for 3 minutes and add 3 mL DMEM to each sample.

Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant leaving ~0.5mL.

Resuspend cell pellet and transfer to labeled 10cm plate with 10 mL Fibroblast media for each sample.

Place 10 cm plates in 37C/5% CO2 incubator.

Change media on the cells after 18-24 hours and change the media every 3-4 days until confluent using Fibroblast media.

Harvest cells by removing media and adding 2 mL TrypLE to 10 cm plate. Incubate at room temperature until cells begin to detach.

Resuspend cells and transfer to 15 mL tube with 2 mL Fibroblast media.

Centrifuge cells at 1500 rpm for 3 minutes. Remove supernate and resuspend cells in residual volume.

Transfer 1/10 of cells to new 10 cm plate with 10 mL Fibroblast media to continue to growing.

Freeze down the remainder of the cells in fibroblast freezing media (50% FBS/40% Fibroblast media/10% DMSO) in 1 mL aliquots (2-3 aliquots/10cm plate)