Spleens were dissociated with 2 mL of 1× PBS using frosted glass slides. The cells were pelleted by centrifugation at 4 °C for 5 min at 1500 rpm. The pellet was resuspended, and the red blood cells were lysed with 1 mL of H2O followed by the addition of 1 mL of 2× PBS. Large debris was allowed to settle. The supernatant was then transferred to a new tube, which was centrifuged to obtain a cell pellet. The splenocytes were resuspended with 1.5% FBS in 1× PBS for flow cytometry. CT26 tumors from BALB/c mice were dissociated with the GentleMACs dissociator and mouse tumor dissociation kit (Miltenyi, Germany, 130–096–730) following the manufacturer’s protocol.
Gentlemacs dissociator and mouse tumor dissociation kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The GentleMACs dissociator is a laboratory equipment designed for the mechanical dissociation of tissue samples. The mouse tumor dissociation kit is a reagent set used in conjunction with the GentleMACs dissociator for the enzymatic and mechanical dissociation of mouse tumor tissue. The core function of these products is to enable the preparation of single-cell suspensions from tissue samples.
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4 protocols using gentlemacs dissociator and mouse tumor dissociation kit
1
Splenocyte Isolation and Tumor Dissociation
2
Dissociation of Murine Solid Tumours
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Primary tumour epithelial cells were obtained using the gentleMACS Dissociator and Mouse Tumor dissociation kit (Miltenyi Biotec) following the protocol recommended for dissociation of tough tumours. To ensure efficient dissociation, volumes of enzyme D, enzyme R and enzyme A were scaled up according to the size of the tumour piece (100, 50 and 12.5 μl, respectively, per each 0.5 cm3). The optional red blood cell lysis step was included in the procedure. Resulting cells were plated in complete growth medium in two-dimensional (2D) adherent conditions. Cells at passage 0 were used for all the experiments in this study.
3
Dissociation and culture of primary tumor cells
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Primary epithelial cells (from three distinct tumors (namely #1, #2, #3) from each mouse model or from three PDX implants) were obtained using the gentle MACS™ Dissociator and Mouse Tumor dissociation kit (Miltenyi Biotec, Bisley, Surrey, UK) following the manufacturer’s recommendations using the protocol for ‘Dissociation of Tough Tumors’ for mouse tumors and the protocol for ‘Dissociation of Soft and Medium Tumors’ for the PDX. To ensure efficient dissociation volumes of Enzyme D, Enzyme R and Enzyme A were scaled up according to the size of the tumor piece (100 μL, 50 μL and 12.5 μL respectively per each 0.5 cm3). The optional steps - the short spin for collection of the dissociated material at the bottom of the MACS tube and red blood cell lysis - were included in the procedure.
Mouse cells were cultured in complete growth medium in 2D adherent conditions for expansion or in 3D for functional studies. Cells up to passage 5 were used for all the experiments in this study. Freshly isolated human PDX cells were grown in HuMEC Ready Medium (Thermo Fisher Scientific) in Matrigel in 3D. Cultures were maintained at 37°C in a 5% CO2/5%O2 atmosphere in a Galaxy 170R incubator (New Brunswick, Eppendorf).
Mouse cells were cultured in complete growth medium in 2D adherent conditions for expansion or in 3D for functional studies. Cells up to passage 5 were used for all the experiments in this study. Freshly isolated human PDX cells were grown in HuMEC Ready Medium (Thermo Fisher Scientific) in Matrigel in 3D. Cultures were maintained at 37°C in a 5% CO2/5%O2 atmosphere in a Galaxy 170R incubator (New Brunswick, Eppendorf).
4
Dissociation and Analysis of TUBO Tumors
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