Materials, Equipment & Reagents including Formulations
Liberase: (Cat# 5401127001; Roche)
DNase I (Cat# 4380-096-06; R&D)
i) C-tubes for GentleMACS (Cat# 130-093-237, Miltenyi Biotec)
ii) MACS SmartStrainers 70µm (Cat# 130-098-462, Miltenyi Biotec)
iii) Sterile serological pipets; 50, 25,10, 5, 2, 1 mL size
iv) Sterile pipet tips; 1000, 200 and 20 μl
v) Sterile 15mL or 50mL conical polypropylene tubes
vi) Test tube racks for 15mL or 50mL conical tubes
vii) LD columns (Cat# 130-042-901; Miltenyi Biotec)
i) GentleMACS Tissue Octo Dissociator (Miltenyi Biotec Inc San Diego, CA)
iii) Table Top Centrifuge
iv) Biological Safety Cabinet, Class II
v) Dissecting forceps and scalpel
Measure of senescence (C12FDG) by flow cytometry
o10 ug – Sigma Catalog number B1793
-C12FDG – 5 mg – Invitrogen Catalog number D2893
-0.1 mM bafilomycin A1: first reconstitute 100 ug in 160.56 ul DMSO for a final concentration of 1 mM. Use this intermediate dilution to make aliquots at a concentration of 0.1 mM (1 ul + 9 ul DMSO).
-2 mM C12FDG: Dilute 5mg of C12FDG in 0.3 ml of DMSO to make 20mM stock. Dilute the 20 mM stock solution of C12FDG 1:10 with fresh cell culture medium to make a 2 mM working solution just before its addition into the cell culture.
CD326 (Biolegend; Cat# 118213)
CD140a (Invitrogen; Cat# 12-1401-81)
CD31 (Biolegend; Cat# 102528)