Application protocol

Isolation and analysis of tissue-derived
CD11c+ mouse dendritic cells  

This application protocol describes the procedure to dissociate mouse spleen, lung, and intestine using the gentleMACS™ Dissociator and specific tissue dissociation kits in order to obtain viable subsets of DCs that can be further isolated using MACS® Cell Separation Technology or flow sorting.

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Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

General reagent and instrument requirements

  • MACSmix™ Tube Rotator (# 130-090-753) in combination with an incubator at 37 °C
  • gentleMACS™ Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (# 130-093-237, # 130-096-334)
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.

PBE buffer for spleen/lung tissue dissociation and for cell isolation

Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130-091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222). Keep buffer cold (2–8 °C). 
▲ Note: Always use freshly prepared buffer. Do not use autoMACS Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.

For spleen tissue dissociation

  • Spleen Dissociation Kit, mouse (# 130-095-926)
  • Phosphate-buffered saline pH 7.2
  • Pre-Separation Filters, 70 μm (# 130-095-823)

For lung tissue dissociation

  • Lung Dissociation Kit, mouse (# 130-095-927)
  • Phosphate-buffered saline pH 7.2
  • MACS SmartStrainers (70 µm) (# 130-098-462)

For intestine tissue dissociation

  • Lamina Propria Dissociation Kit, mouse (# 130-097-410)
  • MACS SmartStrainers, 100 μm (# 130-098-463)
  • (Sterile) 1× Hank’s balanced salt solution (HBSS) without Ca2+ and Mg2+ containing 10 mM HEPES. Store at room temperature. This solution is referred to as HBSS (w/o) in the protocol of the kit data sheet.
  • (Sterile) 1× HBSS with Ca2+ and Mg2+ containing 10 mM HEPES. Store at room temperature. This solution is referred to as HBSS (w) in the protocol of the kit data sheet.
  • (Sterile) Predigestion solution: prepare 1× HBSS (w/o) containing 5 mM EDTA, 5% fetal bovine serum (FBS), 1 mM DTT freshly before each digestion. Store at room temperature.
    Note: Per digestion a volume of 40 mL of the predigestion solution is required. 
  • (Sterile) Digestion solution: prepare 1× HBSS (w) containing 5% FBS freshly before each digestion. Store at room temperature.
    Note: Per digestion a volume of 2.5 mL of the digestion solution is required. 
  • (Sterile) PB buffer: prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5 % bovine serum albumin (BSA). Keep buffer cold (2–8 °C).
    Note: BSA can be replaced by other proteins such as mouse serum albumin, mouse serum, or FBS. 

For cell isolation and flow cytometry analysis

  • MACSQuant Analyzer 10 (# 130-096-343)
  • MACS Columns and MACS Separators: CD11c+ cells can be enriched using MS, LS, or XS Columns. Positive selection can also be performed on the autoMACS Pro Separator.
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Positive selection
MS1×1072×108MiniMACS™, OctoMACS™,
VarioMACS, SuperMACS II
LS1×1082×109MidiMACS™, QuadroMACS™,
VarioMACS, SuperMACS II
XS1×1092×1010SuperMACS II
autoMACS2×1084×109autoMACS Pro

▲Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet. 

  • (Optional) Fluorochrome-conjugated CD11c antibodies for flow cytometry analysis, e.g., CD11c-VioBlue® (# 130‑102‑413). Learn more about our antibodies and dyes.
  • (Optional) FcR Blocking Reagent, mouse (# 130-092-575) to avoid Fc receptor–mediated antibody labeling 
  • (Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cells
  • (Optional) Dead Cell Removal Kit (# 130-090-101) for the depletion of dead cells
  • (Optional) Pre-Separation Filters, 70 μm (# 130-095-823) to remove cell clumps
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Protocol


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The following protocol summarizes steps to produce single-cell suspensions from mouse spleen, lung and intestine tissue and isolate viable dendritic cells