An In Vitro Method to Identify Innate and Adaptive Immune Cells Residing in Murine Lungs

Published: April 30, 2024

Abstract

Source: Christofides, A., et al. Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung. J. Vis. Exp. (2021).

This video demonstrates an in vitro technique to identify immune cells residing in murine lungs. After isolating target immune cells from murine lung tissue, they are surface-labeled using a cocktail of fluorophore-tagged antibodies against specific markers on innate and adaptive immune cells. Additionally, monocytes, macrophages, and dendritic cells are labeled using an intracellular marker. The various immune cell types are then identified using flow cytometry.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Surgical excision and tissue preparation

  1. Euthanize the mouse by intraperitoneally injecting 1 mL of tribromoethanol (prepared according to standard protocol; Table of Materials).
    NOTE: CO2 asphyxiation should be avoided in lung studies as it might cause lung injury and alter the features and properties of lung immune cells. Cervical dislocation should also be avoided as it might cause mechanical injury of the lung.
  2. Transfer the mouse to a clean and dedicated area for surgical operation.
  3. Stabilize the mouse dorsal side down by using needles or tape on the four extremities. Use 70% ethanol to sanitize the skin of the ventral area.
  4. Perform an incision in the skin, from the neck to the abdomen. Carefully remove the skin from the thoracic area.
  5. Carefully remove the sternum and ribs.
  6. Flush the lungs by injecting 10 mL of cold phosphate-buffered saline (PBS) directly in the right ventricle, using an 18-21 G needle, until the lungs become completely white.
  7. Carefully remove the thymus and heart without touching the lungs.
  8. Gently detach the lungs from the surrounding tissues and transfer them to a tube with cold bovine serum albumin (BSA) buffer (Table 1).
    NOTE: Effort should be made to remove all adjacent fat from the lungs before further preparing the single-cell suspension, as this could bias the readouts.

2. Preparation of single-cell suspension

  1. Transfer the lungs to an empty Petri dish and mince them with two fine scalpels. Transfer all the pieces of the minced lung to a new 50 mL conical tube. Use 5 mL of digestion buffer to wash the plate and add it to the 50 mL tube containing the minced lung (Table 1).
    NOTE: Digestion buffer should be prepared immediately before use. Use 5 mg/mL of collagenase. Combining 1 or 5 mg of collagenase with BSA buffer or protein-free PBS did not improve results (Figure 1).
  2. Secure the lid of the tube and digest the lung for 30 min on an orbital shaker at a speed of 150 rpm at 37 °C. Stop the reaction by adding 10 mL of cold BSA buffer.
  3. After digestion, use an 18 G needle to mix and dissolve the lung pieces. Place a 70 µm filter strainer at the top of a new 50 mL conical tube.
    NOTE: Usage of a smaller micron filter might result in the loss of major myeloid populations.
  4. Slowly transfer the digested lung mixture directly on the strainer. Use the rubber side of a 10 mL syringe plunger to smash the remaining lung pieces on the filter. Wash the processed material on the filter with BSA buffer.
  5. Centrifuge the single-cell suspension at 350 × g for 8 min at 4 °C.
  6. Carefully discard the supernatant and resuspend the cells in 1 mL of ammonium–chloride–potassium (ACK) lysis buffer. Mix well using a 1 mL pipet, and incubate for 90 s at room temperature.
  7. Add 10 mL of cold BSA buffer to stop the reaction and centrifuge at 350 × g for 7 min at 4 °C.Carefully discard the supernatant and resuspend the pellet in Staining Buffer to count the cells using a hemocytometer.
  8. Resuspend the cells at a concentration of 5 × 106 cells/mL and use them for surface staining (see section 2).
    NOTE: For this purpose, plate the cells in a 96-well round-bottom plate followed by antibody staining and washes. If a plate centrifuge is not available, use flow tubes instead of plates. With this protocol, ~15-20 × 106 cells per lung can be obtained from a 6-10-week-old C57BL/6 mouse of average size.

3. Surface antibody staining

  1. Transfer 1 × 106 cells in 200 µL per well in a 96-well plate. Centrifuge the plate at 350 × g for 7 min at 4 °C. In the meantime, prepare the Fc-block solution by diluting anti-16/32 antibody (1:100) in staining buffer (Table 1).
  2. Resuspend the cells in 50 µL of the pre-prepared Fc-blocking solution (Table of Materials) and incubate for 15-20 min at 4 °C or on ice.
  3. Add 150 µL of staining buffer and centrifuge the plate at 350 × g for 5 min at 4 °C. Meanwhile, prepare the surface antibody cocktail by diluting surface antibodies (1:100; Table 2) in staining buffer.
    NOTE: (i) Anti-16/32 antibody for Fc-blocking can be used with the surface antibodies in the same mixture. (ii) If fixable viability dye is used, add it to the surface antibody cocktail at a dilution of 1:1,000.
  4. Resuspend the cells in 50 µL of the pre-prepared surface antibody cocktail and incubate for 30-40 min at 4 °C in the dark. Wash the cells with staining buffer twice.
    ​NOTE: If no intracellular staining is required, resuspend the cells in 200 µL of staining buffer and proceed directly to the acquisition of data on the flow cytometer. Alternatively, cells might be fixed and stored at 4 °C for acquisition later. We recommend using the cells for flow cytometry within 24 h.

4. Cell fixation and intracellular staining

  1. Prepare the fixation/permeabilization buffer (Fix/Perm Buffer) by mixing three parts of fixation/permeabilization concentrate and 1 part of fixation/permeabilization diluent of the FoxP3/Transcription Factor Staining Buffer Set (Table 1).
  2. Resuspend the cells in 50 µL of the pre-prepared Fix/Perm Buffer per well of the 96-well plate, where cells were plated as described in section 3, and incubate them for 20-25 min at 4 °C in the dark.
  3. Dilute the 10x permeabilization buffer as 1: 10 in purified deionized water to prepare 1x permeabilization buffer.
  4. Wash the cells once with 1x permeabilization buffer. Meanwhile, prepare the intracellular antibody cocktail by diluting intracellular antibodies (1:100) in 1 mL of permeabilization buffer.
  5. Resuspend the cells using 50 µL of the pre-prepared surface antibody cocktail per cell of the 96-well plate and incubate for 40 min at 4 °C in the dark.
  6. Wash the cells once with permeabilization buffer and once with staining buffer. After the final wash, resuspend the cells in 200 µL of staining buffer.
    NOTE: If no flow cytometer with plate reader is available, transfer the cells into flow cytometry tubes.
  7. Acquire a minimum of 1.5 × 106 cells per sample on the flow cytometer.
    NOTE: For single colors and unstained control samples, 0.5-1 × 106 cells per sample will be sufficient. It is recommended to titer the individual antibodies used to achieve optimal staining and reduce costs. The present protocol has been optimized using Fix/Perm Buffer prepared using the FoxP3 staining buffer set. Because CD68 is a cytoplasmic and not a nuclear marker, other permeabilization solutions such as a low concentration of paraformaldehyde or cytofix/ cytoperm kits from various vendors might be sufficient.

Table 1: Buffers.

BSA buffer PBS + 0.5% Bovine serum albumin
Digestion buffer Prewarmed (37 °C) BSA buffer + 5 mg/mL collagenase type 1 + 0.2 mg/mL DNase I
Staining buffer PBS + 2.5% FBS
Fix/Perm Buffer Three parts of fixation/permeabilization diluent and 1 part of fixation/permeabilization diluent of the Foxp3/Transcription Factor Staining Buffer Set
Permeabilization buffer 10x permeabilization buffer from the Foxp3/Transcription Factor staining Buffer Set diluted 10 times in purified deionized water

Table 2: Usage of monoclonal antibodies.

Antigen Clone Fluorochrome Dilution Surface/intracellular
CD45 30-F11 APC/CY7 1:100 surface
Gr-1 RB6-8C5 BV421 1:100 surface
CD68 FA-11 PerCPCy5.5 1:100 intracellular
CD11b M1/70 PECy7 1:100 surface
Siglec F S17007L FITC 1:100 surface
CD11c N418 BV650 or BV510 1:100 surface
CD64 X54-5/7.1 PE/Dazzle 594 1:100 surface
MHC-II M5/114.15.2 AF700 1:100 surface
CD103 2.00E+07 PE / FITC 1:100 surface
Live/Dead Fixable Far Read Dead Cell Stain Kit FarRed (APC) or Aqua (BV510) 1:1000 surface
CD3 17A2 PE 1:100 surface
B220 RA3-6B2 AF488 1:100 surface
NK1.1 PK136 FITC 1:100 surface
CD24 30-F1 PE 1:100 surface
MERTK 2B10C42 PE 1:100 surface
F4/80 BM8 BV605 1:100 surface
CX3CR1 SA011F11 PE 1:100 surface
FcBlock (CD16/32) 93 1:100 surface

Representative Results

Figure 1
Figure 1: The best cellular dissociation is achieved by 5 mg/mL of collagenase 1 in prewarmed PBS + 0.5% BSA. In all different conditions, 0.2 mg of DNAse I was also included. Abbreviations: BSA = bovine serum albumin; FSC-A = peak area of forward-scattered light; L/D = live/dead staining; SF = Siglec F.

Divulgaciones

The authors have nothing to disclose.

Materials

10 mL syringe plunger EXELINT 26265
18 G needles BD Precision Glide Needle 305165
21 G needles BD Precision Glide Needle 305195
50 mL conical tubes Falcon 3520
70 μm cell strainer ThermoFisher 22363548
96-well plates Falcon/corning 3799
ACK Lysing Buffer ThermoFisher A10492-01
anti-mouse CD11b Biolegend 101215 For details see Table 2
anti-mouse CD11c Biolegend 117339 / 117337 For details see Table 2
anti-mouse CD45 Biolegend 103115 For details see Table 2
anti-mouse CD64 Biolegend 139319 For details see Table 2
anti-mouse CD68 Biolegend 137009 For details see Table 2
anti-mouse GR-1 Biolegend 108433 For details see Table 2
anti-mouse Siglec F Biolegend 155503 For details see Table 2
AVERTIN Sigma-Aldrich 240486
B220 Biolegend 103228 For details see Table 2
Bovine Serum Albumin (BSA) Sigma-Aldrich 9048-46-8
CD103 Biolegend 121405 / 121419 For details see Table 2
CD24 Biolegend 138503 For details see Table 2
CD3 Biolegend 100205 For details see Table 2
Centrifuge
Collagenase Type 1 Worthington Biochemical Corp LS004196
CX3CR1 Biolegend 149005 For details see Table 2
DNase I Millipore Sigma 10104159001
Ethanol
F4/80 Biolegend 123133 For details see Table 2
FcBlock (CD16/32) Biolegend 101301 For details see Table 2
Fetal Bovine Serum R&D Systems
Fine Serrated Forceps Roboz Surgical Instrument Co
Foxp3 / Transcription Factor Staining Buffer Set ThermoFisher 00-5523-00
Futura Safety Scalpel Merit Medical Systems SMS210
Live/Dead Fixable Far Read Dead Cell Stain Kit ThermoFisher L34973 For details see Table 2
MERTK Biolegend 151505 For details see Table 2
MHC-II Biolegend 107621 For details see Table 2
NK1.1 Biolegend 108705 For details see Table 2
Orbital Shaker VWR Model 200
Petri dish Falcon 351029
Refrigerated benchtop centrifuge SORVAL ST 16R
Small curved scissor Roboz Surgical Instrument Co

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An In Vitro Method to Identify Innate and Adaptive Immune Cells Residing in Murine Lungs. J. Vis. Exp. (Pending Publication), e22141, doi: (2024).

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