Hackstein1 project protocol

Respiratory dendritic cell subsets and lymphocyte populations in 5 inbred strains of mice   (2012)

Hackstein H
With: Wachtendorf A, Kranz S, Lohmeyer J, Bein G, Baal N

Hackstein1 Protocol

Project protocol - Contents

Workflow and sampling

Data collected
Cohort 1: Lung single-cell suspensions prepared and analyzed Flow cytometer
Dendritic cell counts, white blood cell counts (subsets)
Cohort 2: Bronchoalveolar lavage preformed and cells analyzed Flow cytometer
Total leukocytes, percent macrophages

Equipment and supplies

  • Syringes and needles
  • Dissection kit
  • 20 G 1 1/2 canules (0.9 x 40 mm) (BD GERMANY)
  • Cell strainer 70 µM (BD GERMANY)
  • FACS Canto II flow cytometer (Becton Dickinson, San Jose CA)

Reagents and solutions

  • Type A collagenase (Roche GERMANY)
  • DNase (Roche GERMANY)
  • Fetal calf serum (FCS) (PAA GERMANY)
  • Ammonium chloride
  • PBS, 5 mM EDTA (pH 7.2)
  • Permeabilization buffer (ebioscience GERMANY)
  • Fluorochrome-labeled monoclonal antibodies (for full description see Table 1, Hackstein et al., 2012)

Procedure: Lung preparation

    1. Mice are euthanized, and lungs are perfused via the right ventricle with HBSS to remove intravascular cells.
    2. Lung tissue is minced and digested in 0.09 U/mL type A collagenase and 9.09 U/mL DNase in IMDM with 10% FCS at 37°C for 1h.
    3. Single-cell suspensions are prepared by tissue resupension with canules and by mashing through a 70 µM cell strainer.
    4. Red blood cells are lysed by ammonium chloride.
    5. Cells are washed with HBSS for flow cytometry (see below).

Procedure: Bronchoalveolar lavage (BAL)

    1. Mice are euthanized and the trachea exposed.
    2. A small incision is made in the trachea to insert a shortened 21-gauge cannula that is firmly fixed and connected to a 1-mL syringe filled with 300 µL of PBS, 5 mM EDTA (pH 7.2).
    3. BAL is performed with 300 µL aliquots until a BAL volume of 1.5 mL is recovered.
    4. Cells are separated by centrifugation and are resuspended for flow cytometry (see below).

Procedure: Flow cytometry

    1. Fluorochrome-labed monoclonal antibodies conjugated to FITC, PE, PeCy7, PerCPCy5.5, APC, APC-Cy7, Pacific Blue, and appropriate isotype controls are used for surface staining according to manufacturer's instructions (full descriptions of monoclonal antibodies, fluorochrome, and source are given in Table 1, Hackstein et al., 2012).
    2. Cells are stained for 30 min on ice.
    3. Cells are washed with staining buffer (PBS, 5% FBS) at room temperature.
    4. Cellular phenotyping is performed on a FACS Canto II flow cytometer (gating strategies are well documented in Hackstein et al., 2012)

Data collected by investigator

  • BAL leukocytes
  • BAL percent macrophages
  • Lung macrophages
  • Lung neutrophils
  • Lung eosinophils
  • Lung dentritic cells and subsets
  • Lung classical lymphocytes
  • Lung innate lymphocytes
  • Lung Linneg leukocytes
  • Lung T regulatory cell populations