For all three human GBMs (6969, GS2, 7192), we dissociated 1 × 106 tumor cells using Accutase, washed cells twice in AutoMACS rinsing buffer (Miltenyi Biotech.), and then incubated human GBM cells with rat PDPN-PE (1:50, Angiobio) or CD133-PE (1:20, clone 293C, Miltenyi Biotech.) antibodies for 45 min at 4°C in rinsing buffer and FCR human blocking reagent (1:10, Miltenyi Biotech.). The cells were washed three times in rinsing buffer and incubated with microbead-tagged anti-PE antibodies (Miltenyi Biotech.) at 4°C in rinsing buffer with FCR human blocking reagent for 30 min. After three washes, we suspended cells in 500 ml rinse buffer and separated by MACS using LS positive selection columns (Miltenyi Biotech.). The negative (CD133- or PDPN-) fraction was depleted from remaining positive cells using a 2nd round of MACS. Self-renewal capacity was assayed as mentioned above.
Automacs rinsing buffer
Manufactured by Miltenyi Biotec
Sourced in Germany
The AutoMACS rinsing buffer is a buffer solution designed for use with the AutoMACS Pro Separator, a magnetic cell separation instrument manufactured by Miltenyi Biotec. The buffer is used to rinse and maintain the system during operation.
Automatically generated - may contain errors
Lab products found in correlation
Human fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Cd133 pe, by Miltenyi Biotec (1 mentions)
Ls positive selection column, by Miltenyi Biotec (1 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Ficoll isopaque lymphoprep, by Axis-Shield (1 mentions)
Anti clumping agent, by Thermo Fisher Scientific (1 mentions)
3 protocols using automacs rinsing buffer
1
Enrichment and Isolation of GBM Cell Populations
2
Isolation of Pan-T Cells from PBMCs
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Peripheral blood mononuclear cells (PBMCs) were obtained from sodium-heparin-anticoagulated blood by Isopaque-Ficoll (Lymphoprep; Axis-Shield, Oslo, Norway) gradient centrifugation. PBMCs were then resuspended in autoMACS® rinsing buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) for negative selection of pan-T cells with Pan-T cell Isolation Kit (Miltenyi Biotec) following manufacturer's instructions. Pelleted T cells were stored at -80 °C before RNA isolation.
3
Senescence Assay of Cryopreserved BM Samples
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Six LR-MDS and six HR-MDS cryopreserved BM samples were thawed and washed in PBS with anti-clumping agent according to the manufacturer’s instructions (Gibco, Waltham, MA, United States). The cells were washed twice in autoMACS rinsing buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated for 1 hour (37 °C, 5% CO2) with the β-galactosidase stain FITC (Senescence assay kit; Abcam, Cambridge, UK). Then, the cells were washed in PBS and stained for 30 min in a cocktail of antibodies specified in the Supplementary Methods . The cells were washed and directly measured at Cytek Aurora (Cytek, Fremont, CA, USA). The data were analyzed with the FlowJo software (BD, Franklin Lakes, NJ, USA).
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