Tumor dissociation into single-cell suspension

Isolation of primary tumour cells into single cell suspensions

1.      Remove mammary tumours from mice.

2.      Mince tissue with razor blades on aseptic surface.

3.       Dissociate cells by incubating in F12/DMEM (1:1) with 5% FBS, 20ug/ml gentamycin, 300units/ml collagenase and 100units/ml hyaluronidase at 37⁰C for 2 hours. 

4.      Centrifuge cells at 400g for 10 mins and wash with PBS.

5.      Incubate cells for another 5 mins in 0.05% trypsin/0.025% EDTA.

6.      Add an equal volume of F12/DMEM with 5% FBS + 2units/ml DNAseI.

7.      Pass cells through a 40um nyon mesh filter.

8.      Centrifuge at 400g for 5 mins.

9.      Resuspend cells with red blood cell lysis buffer for 5 mins at RT.

10.   Centrifuge cells at 400g for 5 mins and resuspend in F12/DMEM with 10%FBS, 5ug/ml insulin, 10ng/ml EGF and Pen/Strep or FACS buffer for subsequent flow analysis. (For scRNAseq and subsequent Chromium 10x steps, resuspend in PBS with 0.04% BSA).