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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The protocol described below is a simple and effective way to isolate retinoid-containing cells from highly heterogeneous lung cell populations by making use of specific retinoid autofluorescence and by employing fluorescent-activated cell sorting.

Abstract

Retinoids (vitamin A and its metabolites) are an essential lipid component of the alveolar microenvironment, and cell-type specific retinoid metabolism is required to maintain the functional health of the developing and adult lungs. Lung cells utilize specific pathways, allowing for the efficient uptake of circulating retinoids from the blood as retinol (ROH), followed by intracellular stepwise conversion of ROH into the transcriptionally active retinoid species, all-trans-retinoic acid (ATRA). ATRA-mediated (or retinoid-mediated) signaling is crucial for regulating lung alveolarization, surfactant production, angiogenesis, permeability, and immunity. Importantly, specific lung cells, including fibroblasts, can accumulate retinoids in the form of retinyl esters (RE), which can be stored or further mobilized as ROH for transfer to the neighboring cells when needed. Lung retinoid-containing cells can be isolated and collected from the single-cell suspension of digested lungs by making use of retinoid autofluorescence (the emission at 455 nm upon excitation at 350 nm) and by employing fluorescence-activated cell sorting (FACS). Additional cell-specific in vivo labeling of lung cells with red fluorescent protein allows isolating and collecting specific retinoid-containing lung cell populations. The collected cells can be directly analyzed or cultured for further analyses of cell morphology, gene expression, and responsiveness to pharmacological manipulations. This technique of isolation and application is important for animal model studies of lung health and lung injury to gain deeper insight into cellular aspects of retinoid metabolism in the lungs and lipid-mediated cellular communications.

Introduction

Retinoids (vitamin A and its metabolites) are an essential lipid component of the alveolar microenvironment, and cell-type specific retinoid metabolism and signaling are required to maintain the functional health of the developing and adult lung1,2,3,4,5,6,7,8,9,10,11,12,13,14. Lung cells utilize specific pathways, allowing for the efficient uptake of circulating dietary-derived retinoids from the blood as retinol (ROH)15,16,17,18,19, followed by intracellular stepwise conversion of ROH into the transcriptionally active retinoid species, all-trans-retinoic acid (ATRA)20. ATRA-mediated (or retinoid-mediated) signaling is achieved through ATRA interaction with its three distinct cognate nuclear hormone receptors, retinoic acid receptors (RARα, RARβ, and RARγ21,22), and is crucial for regulating lung alveolarization23,24,25,26,27,28,29, surfactant production30,31,32,33,34,35, angiogenesis36, permeability37, and immunity38,39,40. Importantly, specific lung cells, especially lung fibroblasts, can accumulate retinoids in the form of retinyl esters (RE), which can be stored or further mobilized as ROH for transfer to the neighboring cells when needed1.

The complexity of retinoid metabolism and signaling, as well as the cellular complexity of the lung, make studies aimed at exploring retinoid metabolism in the lung in vivo challenging. We have outlined a simple and robust protocol for isolating retinoid-containing cells (Figure 1) from highly heterogeneous lung cell populations by making use of specific retinoid autofluorescence (the emission at 455 nm upon excitation at 350 nm) and by employing fluorescence-activated cell sorting (FACS). The protocol does not require additional cell labeling except for viability staining if the goal of the study is to isolate and characterize primary live lung cells based on their ability to store retinoids. This significantly reduces the preparation time for cell sorting, eliminates the need for additional staining, and allows for isolating high yields of viable primary cells. However, if the goal of the study is to isolate and characterize specific lung cell populations (fibroblasts, endothelial, epithelial, or immune cells), additional cell sorting can be performed after labeling the sorted retinoid-containing cells with cell-specific antibodies.

Retinoid autofluorescence has been used in published studies to establish the identities of retinoid-containing cells and/or to quantify the abundance of these cells in the liver41,42,43,44, pancreas45,46, kidneys41,47, and lung41. Moreover, several research groups reported the use of retinoid fluorescence to isolate by FACS and study primary retinoid-containing cells from living tissues, including liver44,48,49,50,51,52,53 and lung1. In the current protocol, we show how specific cell populations can be labeled in vivo prior to isolating retinoid-containing cells using tdTomato (red fluorescent protein). tdTomato's spectral characteristics (the emission at 581 nm upon excitation at 554 nm54) and brightness do not interfere with retinoid autofluorescence and, therefore, make it convenient to achieve cell specificity during sorting. Given the critical role of uncompromised retinoid metabolism and signaling within the normal alveolar microenvironment1, the described technique of lung cell isolation is a useful tool in animal model studies of lung health and disease to gain deeper insight into cellular aspects of retinoid metabolism in the lungs and lipid-mediated cellular communications in vivo.

Protocol

All described procedures and experiments involving mice were carried out with the approval of the Institutional Animal Care and Use Committee (IACUC) of Rutgers University (IACUC ID: PROTO202200111) according to criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences55.

1. Considerations and preparations for the experiment

  1. Animal husbandry and manipulations
    NOTE: Three-month-old (10-12 weeks-old) male and female mice can be used in the studies. Lecithin:retinol acyltransferase-deficient mice (Lrat-deficient, Lrat-/- mice56) on a C57BL/6J genetic background and age-matched littermates (wild type, Lrat+/+ mice) were used in the described experiments. Mice expressing the tdTomato gene in fibroblasts (F-tdT mice) were generated by crossing mice harboring a tdTomato reporter cassette (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, Jackson Lab strain #007914) with Col1a2-CreER mice (B6.Cg-Tg(Col1a2-Cre/ERT,-ALPP)7Cpd/2J, Jackson Lab strain #029567).
    1. Employ the Cre-expressing mouse model, one of the many available options to label the target cell population in vivo.
      NOTE: Depending on the choice of the target cell, Cre expression, the efficiency of Cre-mediated recombination, and cell specificity, the investigators may utilize any reliable Cre-expressing mouse model.
    2. Use tdTomato transgene expression (upon tamoxifen treatment as described below) to label Col1a2+ fibroblasts, followed by their isolation from single cell suspensions of the digested lung as described below.
  2. Cre-mediated recombination and transgene activation in vivo
    1. Induce Cre expression in F-tdT mice through intraperitoneal (IP) injection of 2 mg of tamoxifen once every 24 h for a total of 5 consecutive days.
    2. Sanitize the injection site with 70% ethanol prior to injection. Use the mice for experiments 1 month after the final tamoxifen injection.
    3. Check the effectiveness of Cre-recombination prior to the sorting experiments. Confirm the effective Cre recombination and tdTomato expression by isolating the target cell population of lung fibroblasts from the tamoxifen-treated F-tdT mice using anti-Pdgfrα magnetic beads and magnetic-activated cell sorting (MACS).
  3. Preparation of solutions, plastic- and glassware
    NOTE: Aseptic techniques should be followed for the procedures described below. The procedures involving solution preparations, tissue digestion, and cell isolation should be done under the laminar hood. Solutions should be sterilized by filtration using a 0.2 µm filter; surgical tools as well as labware should be sterilized by autoclaving.
    1. Under the laminar hood, prepare Hanks' balanced salt solution (HBSS), calcium- and magnesium-free (HBSS without Ca2+/Mg2+, containing 5.3 mM KCl, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 137.9 mM NaCl, 0.3 mM Na2HPO4, 5.6 mM D-Glucose) for the lung perfusion (5 mL per mouse). Sterilize the solution using a 0.2 µm filter and fill up the syringe with the solution.
    2. Under the laminar hood, prepare Hanks' balanced salt solution with calcium- and magnesium (HBSS with Ca2+/Mg2+, containing 1.3 mM CaCl2, 0.5 MgCl2·6H2O, 0.4 mM MgSO4·7H2O, 5.3 mM KCl, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 137.9 mM NaCl, 0.3 mM Na2HPO4, 5.6 mM D-Glucose), containing 0.3 mg/mL of type IV collagenase and 1 mg/mL of dispase for the lung perfusion (10 mL per mouse). Sterilize the solution using a 0.2 µm filter and fill up the syringe with the solution.
    3. Under the laminar hood, prepare HBSS with Ca2+/Mg2+, containing 0.3 mg/mL of type IV collagenase, 1 mg/mL of dispase, and 5 mg/mL of DNase I for lung digestion (10 mL per mouse). Sterilize the solution using a 0.2 µm filter and fill up the syringe with the solution.
    4. Under the laminar hood, fill up a cell culture dish (35 mm 10 mm, one dish per collected lung from one mouse) with 2 mL of sterile HBSS with Ca2+/Mg2+. Place the dish(es) containing HBSS with Ca2+/Mg2+ on ice.

2. Lung perfusion, digestion, and collection of single-cell suspension

  1. Anesthetize the mouse by administering an anesthesia cocktail containing 10 mg/mL ketamine and 2 mg/mL xylazine at a dose of 0.01 mL/g body weight via IP injection.
  2. Make sure that the mouse is in a deep state of unconsciousness by assessing a toe pinch response. Remove the loose hair and visible dirt/debris from the surgical site, clip and wipe clean the surgical site using 70% alcohol for disinfection.
  3. Using sterile surgical tools, open the abdominal and chest cavities. Cut the ribs and the diaphragm to expose the lungs and the heart.
    NOTE: This will assure an instantaneous death as the thoracotomy will cause a cessation of breathing while the mouse is anesthetized; if properly performed, this part of the procedure will take up to 3 min from the start of the procedure till the thoracotomy, followed by the death of the animal.
  4. Cut the inferior vena cava and apply an absorbent pad to absorb the released blood.
  5. Perfuse the lungs in situ through the heart's right ventricle using a 10 mL syringe with a 25 G X 1" needle filled with 5 mL of sterile HBSS without Ca2+/Mg2+. If the perfusion is successful, the lung tissue will become white.
  6. Perfuse the lungs in situ using a 10 mL syringe with a 25 G X 1" needle filled with 10 mL of sterile HBSS with Ca2+/Mg2+, containing type IV collagenase (0.3 mg/mL) and dispase (1 mg/mL).
  7. Remove the lungs and transfer them into a cell culture dish (35 mm X 10 mm) with 2 mL of sterile HBSS with Ca2+/Mg2+.
  8. Under the laminar hood, rinse the lungs and mince them into small pieces using a sterile surgical blade (#20, fits handle #4). Transfer the minced lung into a 15 mL tube by adding the first 5 mL of HBSS with Ca2+/Mg2+ containing 0.3 mg/mL of type IV collagenase, 1 mg/mL of dispase, and 5 mg/mL of DNase I to the minced tissue and transferring it using a 10 mL serological pipette. Collect and transfer the remaining minced tissue residues by washing the cell culture dish with the remaining 5 mL of HBSS with Ca2+/Mg2+ containing 0.3 mg/mL of type IV collagenase, 1 mg/mL of dispase, and 5 mg/mL of DNase I.
  9. Incubate the minced lung tissue in HBSS with Ca2+/Mg2+ containing 0.3 mg/mL of type IV collagenase, 1 mg/mL of dispase, and 5 mg/mL of DNase I on a rotating shaker maintained at 37 °C for 45 min. At each of the three 15-min intervals, under the laminar hood, pass the minced tissue 10 times through a 10 mL serological pipette to better dissociate cells.
  10. Pass the resultant cell suspension through a 100 µm strainer to collect single cells into a 50 mL tube containing 20 mL of cold live cell imaging solution (physiological saline containing 20 mM HEPES, pH 7.4) containing less than 5% of fetal bovine serum (FBS). Collect the cells by centrifugation at 500 g for 10 min at 4 °C.
  11. Aspirate the supernatant and resuspend the cell pellet in 1 mL of red blood cell lysing buffer and leave the tube for 5 min at room temperature (RT) to eliminate erythrocytes. Add 20 mL of cold live cell imaging solution containing less than 5% FBS. Collect the cells by centrifugation at 500 g for 10 min at 4 °C.
  12. Aspirate the supernatant and resuspend the cell pellet in 20 mL of cold live cell imaging solution containing less than 5% FBS. Pass the resultant cell suspension through a 40 µm strainer to collect single cells into a 50 mL tube containing 20 mL of cold live cell imaging solution containing less than 5% FBS. Collect the cells by centrifugation at 500 g for 10 min at 4 °C.
  13. Aspirate the supernatant and resuspend the cell pellet in 10 mL of cold live cell imaging solution containing less than 5% FBS. Count the cells and adjust cell concentration to ~5-10 x 106 cells/ mL.

3. Isolation of retinoid-containing lung cells using Fluorescence Activated Cell Sorting (FACS)

  1. Take an aliquot of cell suspension and set it aside to be used as an unstained gating control. Add viability dye SYTOX Green to the remaining cell suspension (1:1000 dilution, final concentration 30 nM).
  2. Pass cell suspensions through the filter attached to the polystyrene 5 mL (12 x 75 mm) collecting tube.
  3. Proceed with FACS cell isolation by sorting live, individual retinoid-containing cells based on their emission at 455 nm. Perform sequentially singlet discrimination using a plot for forward scatter (forward scatter height/FSC-H versus forward scatter area/FSC-A). Exclude dead cells by scatter characteristics (side scatter) and staining with SYTOX Green.
  4. Gate, sort, and collect single, live, retinoid-containing cells using emission at 455 nm upon excitation at 350 nm.
    NOTE: Using the described procedure, about 5·105 retinoid-containing lung cells can be collected from one mouse lung.
  5. Perform FACS data analysis using flow cytometry software.

Results

Isolation of lung retinoid-containing cells
Mouse lungs (from Lrat+/+ and Lrat-/-) mice were enzymatically digested, and single-cell suspensions were prepared and subjected to FACS according to the procedure outlined above. Cell sorting and data acquisition were performed on a Cytek Aurora™ Cell Sorter System operated by SpectroFlo CS software version 1.3.0 using a 100 µm nozzle and 15 psi pressure. First...

Discussion

The ability of vitamin A detection in human and animal tissues and its histologic visualization using fluorescent microscopy was first reported as early as the 1940s59,60. The phenomenon of retinoid autofluorescence was then successfully applied to studies aimed at locating high concentrations of vitamin A in tissues in vitro61 and characterizing the morphogenesis of animal embryonic tissues62 using fluores...

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was funded by a grant from the National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI) R01 HL171112 (to I.S.), a career development award (to I.S) from Rutgers Center for Environmental Exposures & Disease funded by the National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS) P30 ES005022, and start-up funds from Rutgers, The State University of New Jersey (to I.S.). The authors would like to acknowledge the staff of the Immune Monitoring and Flow Cytometry Shared Resource at the Rutgers Cancer Institute (supported, in part, with funding from the NCI-CCSG P30CA072770-5920) for their contributions to the work presented in this manuscript.

Materials

NameCompanyCatalog NumberComments
10 mL serological pipetteAvantor/VWR76452-284
100 µm strainer Greiner Bio-One542000
15 mL falcon tubeCorning352099
40 µm strainer Greiner Bio-One542040
50 mL falcon tubeCorning352070
Cell culture dish, 35 mm ´ 10 mmCorning430165
Cell sorting mediaGibcoA59688DJ
Collagenase type IV Worthington Biochemical CorporationLS004188 
Cytek Aurora Cell Sorter System Cytek Biosciences
Dispase IISigma-AldrichD4693
DNase ISigma-AldrichDN25
Falcon brand 5-ml polypropylene round bottom tube, 12 mm ´ 75 mmCorning352063
Falcon brand 5-ml polystyrene round-bottom tube with cell-strainer cap, 12 mm ´ 75 mmCorning352235
FlowJo software Becton Dickinsonflow cytometry software
HBSS with Ca2+/Mg2+Gibco14925-092
HBSS without Ca2+/Mg2+Gibco14175-095
Nalgene Rapid-Flow Sterile Disposable Bottle Top FiltersThermoFisher595-3320
Red Cell lysis bufferSigma-AldrichR7757
SpectroFlo CS software Cytek BiosciencesVersion 1.3.0 
Surgical Design Royaltek Stainless Steel Surgical Scalpel BladesFisher Scientific22-079-683
SYTOX Green dead cell stainInvitrogenS34860
TamoxifenSigma-AldrichT2859

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