Dissociation of Tumor Tissue for Flow Cytometry

Single-cell suspensions from tumors were prepared as described previously (20 (link)). Briefly, tumors were isolated, minced, and incubated for 30 to 45 minutes at 37°C in a dissociation solution containing Hanks Balanced Salt Solution supplemented with 0.5 mg/mL collagenase IV (Sigma), 0.1 mg/mL hyaluronidase V (Sigma), and 0.005 MU/ml DNAse I (Sigma). The undigested tissues were removed by passing through 70 μm nylon mesh and centrifuged at 1,500 rpm for 5 minutes. The red blood cells were lysed using RBC lysis buffer (Pharm Lyse; BD Biosciences) and cells were prepared for flow cytometry.

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Rohila D., Park I.H., Pham T.V., Weitz J., Hurtado de Mendoza T., Madheswaran S., Ishfaq M., Beaman C., Tapia E., Sun S., Patel J., Tamayo P., Lowy A.M, & Joshi S. (2023). Syk Inhibition Reprograms Tumor-Associated Macrophages and Overcomes Gemcitabine-Induced Immunosuppression in Pancreatic Ductal Adenocarcinoma. Cancer Research, 83(16), 2675-2689.

Publication 2023

collagenase IV hyaluronidase V DNAse I Pharm Lyse

Buffer Cells Collagenase Dnase Flow cytometry Hanks balanced salt solution Hyaluronidase Ml iv Nylon Tissues Tumors

Corresponding Organization :

Other organizations : University of California, San Diego

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Variable analysis

independent variables

Dissociation solution (containing Hanks Balanced Salt Solution, 0.5 mg/mL collagenase IV, 0.1 mg/mL hyaluronidase V, and 0.005 MU/ml DNAse I)

dependent variables

Cell suspension preparation for flow cytometry

control variables

Tumor isolation and mincing
Incubation time (30 to 45 minutes)
Incubation temperature (37°C)
Removal of undigested tissues by passing through 70 μm nylon mesh
Centrifugation at 1,500 rpm for 5 minutes
Lysis of red blood cells using RBC lysis buffer

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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