LN (pooled inguinal, axillary, brachial, cervical and mesenteric) were digested for 45–60 minutes at 37°C using 0.42 U/mL Liberase TM (Roche) and 60 U/mL DNase I (Sigma) in DMEM (Cellgro) with 2% FCS, essential and non-essential amino acids, sodium pyruvate and HEPES. Red blood cells were lysed (Sigma). CD45+ cells were labeled with CD45 magnetic beads in AwesomeMACS (PBS with 0.5% BSA, 2mM EDTA, 2 mM L-glutamate, 10 mM sodium pyruvate, 1× essential and non-essential amino acids, and 4.5 g/L dextrose) and separated using the Deplete AutoMACS protocol (Miltenyi Biotec). For in vitro co-cultures, DC and macrophages were enriched using CD11c+ and CD11b+ beads (Miltenyi Biotec) prior to CD45 depletion. Cells were stained analyzed by flow cytometry (see below), or sorted by flow cytometry using Influx (BD), FACSVantage (BD) or iCyt Reflection (Sony) cell sorters for in vitro co-cultures and qPCR.
Deplete automacs protocol
Manufactured by Miltenyi Biotec
The Deple AutoMACS protocol is a tool designed for the magnetic separation and depletion of target cell populations from complex biological samples. It utilizes the AutoMACS Pro Separator, a fully automated magnetic cell separation system, to efficiently isolate cells of interest. The core function of this protocol is to provide a standardized and reproducible method for the depletion of unwanted cell types, enabling the enrichment of the desired cell population for further analysis or downstream applications.
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2 protocols using deplete automacs protocol
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Isolation and Enrichment of Immune Cells
2
Isolation and Purification of Immune Cells
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LNs (pooled inguinal, axillary, brachial, cervical and mesenteric) were digested for 45–60 min at 37 °C using 0.42 U ml−1 Liberase TM (Roche) and 60 U ml−1 DNase I (Sigma) in DMEM (Cellgro) with 2% FCS, essential and non-essential amino acids, sodium pyruvate and HEPES. Red blood cells were lysed (Sigma). CD45+ cells were labelled with CD45 magnetic beads in AwesomeMACS (PBS with 0.5% BSA, 2 mM EDTA, 2 mM L-glutamate, 10 mM sodium pyruvate, 1 × essential and non-essential amino acids, and 4.5 g l−1 dextrose) and separated using the Deplete AutoMACS protocol (Miltenyi Biotec). For in vitro co-cultures, DCs and macrophages were enriched using CD11c+ and CD11b+ beads (Miltenyi Biotec) before CD45 depletion. Cells were stained andanalysed by flow cytometry (see below), or sorted by flow cytometry using Influx (BD), FACSVantage (BD) or iCyt Reflection (Sony) cell sorters for in vitro co-cultures and quantitative PCR.
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