Tumor bearing mice were euthanized in accordance with institutional Animal Care and Use Committee approval (#20027-H). PDX were harvested and cut into 2-mm pieces, connective tissue, blood vessels and necrotic tissue was discarded and then transferred into 50-mL Falcon tubes with RPMI, 2% Penicillin/Streptomycin, collagenase 5 (Worthington 1 mg/mL), neutral protease (Worthington 0.5U/ml) and DNAse (Roche, 1 μg/mL), and digested while rotating at 37°C until digestion was complete (Benchmark scientific Roto-therm). All following steps were done on ice or at 4°C. The digested tissue was passed through a 200-μm (Pluriselect) strainer using a syringe plunger for remaining pieces, and then through a 100-μm strainer (Fisher). The cells were spun down at 321×g for 5 minutes at 4°C and the pellet depleted of red blood cells by a 10-second exposure to 1 mL of water followed by the addition of 49 mL of PBS. The cells were counted and plated on collagen coated plates for in vitro experiments. For in vitro experiments, the cells from PDX tumors were subjected to mouse cell depletion according to the manufacturer’s instructions (Miltenyi Biotec).
Mouse cell depletion
Manufactured by Miltenyi Biotec
The Mouse cell depletion equipment is a laboratory tool designed to separate and remove mouse cells from a given sample. It facilitates the isolation and purification of specific cell types by selectively depleting unwanted mouse cells.
Automatically generated - may contain errors
Lab products found in correlation
Dnase, by Roche (4 mentions)
Neutral protease, by Worthington (3 mentions)
Collagenase 5, by Worthington (3 mentions)
Roto therm, by Benchmark Scientific (2 mentions)
100 μm strainer, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Worthington (1 mentions)
Collagenase 4, by Worthington (1 mentions)
4 protocols using mouse cell depletion
1
Isolation and Culture of PDX Tumors
2
PDX Tumor Dissociation and Cell Isolation
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Tumor-bearing mice were euthanized in accordance with Institutional Animal Care and Use Committee approval (#20027-H). PDXs were harvested and cut into 2-mm pieces, connective tissue, blood vessels and necrotic tissue was discarded and then transferred into 50-mL Falcon tubes with RPMI, 2% penicillin/streptomycin, collagenase 5 (Worthington 1 mg/mL), neutral protease (Worthington 0.5 U/mL) and DNAse (Roche, 1 μg/mL), and digested while rotating at 37°C until digestion was complete (Benchmark scientific Roto-therm). All following steps were done on ice or at 4°C. The digested tissue was passed through a 200-μm (Pluriselect) strainer using a syringe plunger for remaining pieces, and then through a 100 μm strainer (Thermo Fisher Scientific). The cells were spun down at 321 × g for 5 minutes at 4°C and the pellet depleted of red blood cells by a 10-second exposure to 1 mL of water followed by the addition of 49 mL of PBS. The cells were counted and plated on collagen-coated plates for in vitro experiments. For in vitro experiments, the cells from PDX tumors were subjected to mouse cell depletion according to the manufacturer's instructions (Miltenyi Biotec).
3
Isolation of Tumor Cells from Tissue Samples
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Tumor tissue was cut into 2 mm pieces in RPMI on ice with the connective tissue, blood vessels and blood clots, and necrotic tissue discarded. These pieces were placed into 50 mL Falcon tubes of RPMI (+2% penicillin/streptomycin), collagenase V (Worthington; 1 mg/mL), neutral protease (Worthington; 0.5 U/mL), and DNase (Roche; 1 μg/mL). The tissue was digested while rotating at 37°C (Benchmark Scientific Roto-Therm) until digestion was complete. Digested tissue was strained through a 200 μm strainer (Pluriselect) using a syringe plunger on the remaining undigested pieces. The digested tissue was then pushed through a 100 μm strainer (Thermo Fisher Scientific), and the cells were spun down at 300g for 5 minutes at 4°C. The supernatant was removed, and the pellet was depleted of RBCs via 10-second resuspension in 1 mL of water followed by 49 mL of 1× PBS. The cells were then spun down at 300g for 5 minutes at 4°C, resuspended in RPMI (+2% penicillin/streptomycin), and counted using a hemocytometer. The cells were then used for either a drug response screening, implantation into mice, or for in vitro experiments. Cells derived from PDX and used for in vitro experiments were subjected to mouse cell depletion according to the manufacturer’s instructions (Miltenyi Biotec).
4
Isolation and Purification of PDX Tumor Cells
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