Isolation, Purification, and Comprehensive Flow Cytometry Assessment of Lung Stromal Cells

Abstract

Stromal cells are non‐hematopoietic cells that consist of endothelial cells and various mesenchymal cell populations. The composition of the stromal cell compartment is diverse in different organs. Numerous recent studies demonstrated that the lung environment contains heterogeneous mesenchymal stromal cell populations with distinctive genomic signatures and location preferences. Besides their role in supporting organ structure and remodeling tissue, mesenchymal stromal cells fulfill critical immune functions. These stromal cells show alterations during lung fibrosis and infectious disorders like COVID‐19 or flu infection.

To date, their identification and isolation were challenging, and most information about their heterogeneity was derived from scRNAseq data. In this protocol, we describe an isolation, comprehensive flow cytometry assessment, and purification strategy for murine lung stromal cells. The described method is optimized for minimizing cell death while keeping a high level of cell purity. This protocol can be also used for ex‐vivo analysis of these cells in downstream functional assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Isolation of stromal cells from murine lung tissue

Basic Protocol 2: Flow cytometry assessment of lung stromal populations

Basic Protocol 3: Purification of lung fibroblastic stromal cells

Alternate Protocol: Positive selection of fibroblastic stromal cells

INTRODUCTION

Stromal cells are heterogenous cells of non‐hematopoietic origin including fibroblasts and endothelial cells. In addition to their classical role of providing the structural scaffold of an organ, stromal cells in lymphoid and non‐lymphoid tissues exhibit immunoregulatory functions at multiple levels e.g., orchestrating recruitment and survival of immune cells and instructing cell differentiation and functions (Lukacs‐Kornek, Malhotra et al., 2011; Lukacs‐Kornek & Turley, 2011). The disposition of fibroblastic stromal cells in the lung microenvironment has gained attention, as they play a key role in the development and progression of multiple pulmonary disorders. In COVID‐19 infection, fatal lung pathology was associated with an increased frequency of mesenchymal fibroblasts that failed to repair the damaged lung (Rendeiro et al., 2021). Damage‐responsive fibroblasts also promoted severe lung damage in influenza infection (Boyd et al., 2020). Specific fibroblast subtypes have also been identified to drive fibrosis in animal models and human pulmonary fibrosis (Mayr et al., 2024). Under physiological conditions, fibroblastic stromal cells in the lung are involved in extracellular matrix remodeling and closely interact with alveolar type 1 and type 2 epithelial cells, modulating surfactant production and cellular differentiation within the alveolar space (El Agha & Thannickal, 2023). scRNAseq analyses revealed the complexity of the fibroblastic cell populations including multiple subsets with unique gene expression profiles and location preference (Liu et al., 2021; Mayr et al., 2024; Rendeiro et al., 2021; Sikkema et al., 2023; Tsukui et al., 2020; Xie et al., 2018).

The isolation of lung stromal cells has been challenging as classical isolation techniques used for immune cell recovery need adjustment to allow stromal cell release from the tissue where these cells are tightly embedded in the extracellular matrix scaffold. Murine studies have focused on using collagen‐reporter mice to help with the identification of fibroblastic cells within the lung environment (Tsukui et al., 2020). This is difficult if genetically modified animals need to be compared where such fluorescence reporters are not necessarily present. Additionally, increasing mechanical disruption usually results in decreased cell viability and could affect the recovery of the sensitive fibroblast subsets from the lung, hindering functional downstream analyses. Here, we describe a combination of mechanical and enzymatic disruption of the murine lung where the major fibroblastic stromal subpopulations could be recovered with high viability and used for comprehensive flow cytometry assessment. Additionally, a gentle and fast purification method was developed to isolate lung fibroblasts without the need of a cell sorter for further functional analyses.

CAUTIONS

All animal experiments were conducted in accordance with the approval of the Animal Ethics Committee of the state of North Rhine‐Westphalia, Germany (AZ: 81‐02.04.2021.A221).

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile techniques should be used accordingly.

NOTE: All protocols involving animals must be reviewed and approved by the appropriate Animal Care and Use Committee and must follow regulations for the care and use of laboratory animals.

Basic Protocol 1. ISOLATION OF STROMAL CELLS FROM MURINE LUNG TISSUE

This protocol describes how to isolate stromal cells including fibroblastic stromal cells from murine lung tissue using a multistep workflow illustrated in Figure 1. The method allows the parallel identification and analyses of not only fibroblasts but also endothelial and epithelial cells, which represent the complete stromal compartment of the lung micro milieu. Additionally, this protocol enables simultaneous and reproducible isolation of stromal cells from multiple experimental animals in parallel and thus allows unbiased comparison of different experimental conditions.

Figure 1.

Workflow overview of the isolation (1) and purification (3) of lung stromal cells from mouse tissue. Flow cytometry enables the identification of sub‐populations of lung stromal cells (2).

Materials

• C57Bl/6J mice

• RPMI 1640 Medium (Thermo Fisher Scientific, cat. no. 61870036)

• Ice

• Digestion buffer (see recipe)

• Collection buffer (see recipe)

• ACK lysis buffer (Thermo Fisher Scientific, cat. no. A1049201)

• PBS, 1× (Thermo Fisher Scientific, cat. no. 10010023)

• Trypan blue stain, 0.4% (Thermo Fisher Scientific, cat. no. T10282)

• FBS (Thermo Fisher Scientific, cat. no. 10082147)

• EDTA, 0.5 M (Thermo Fisher Scientific, cat. no. AM9260G)

• HEPES, 1 M (Thermo Fisher Scientific, cat. no. 15630056)

• Collagenase P from Clostridium histolyticum (Merck, COLLP‐RO, cat. no. 11249002001)

• Collagenase II (Thermo Fisher Scientific, cat. no. 17101015)

• DNase I, grade II (Merck, cat. no. 10104159001)

• Eppendorf tubes:

  • 15 mL (Sarstedt, cat. no. 72.706.400)

  • 2 mL (Sarstedt, cat. no. 72.695.400)

• Bacterial Petri dishes, 10 cm (VWR, cat. no. 391‐2002)

• Fine‐tip scissors

• gentleMACS C Tubes (Miltenyi cat. no. 130‐093‐237)

• Water bath, 37°C

• gentleMACS Octo Dissociator with Heaters (Miltenyi, cat. no. 130‐096‐427)

• Polypropylene centrifuge tubes, 15 mL (Greiner Bio‐One GmbH, cat. no. 188271)

• Filter mesh, 100 µm (cut into 1.5 × 1.5–cm cubes and autoclaved; A. Hartenstein GmbH, cat. no. PES3)

• Centrifuge (Eppendorf, cat. no. 5810 R)

• Countess Cell Counting Chamber Slides (Thermo Fisher Scientific, cat. no. C10228)

• Countess II FL Automated Cell Counter (Thermo Fisher Scientific, cat. no. AMQAF1000)

• 1Sacrifice the mouse with the approved euthanasia procedure.
• 2Carefully open the thorax with a midline incision, cut away the diaphragm, and expose the lungs. Cut out both lungs and place them into a 2‐mL Eppendorf tube filled with precooled RPMI 1640 medium.

  RPMI 1640 medium should be precooled on ice before organ extraction.

  If one of the lobes will be used for histology or other purposes, we recommend using the same lobes of the lung for flow cytometry comparison between experimental conditions.

• 3Place the lung tissue in the bacterial petri dish and with fine‐tip scissors, cut it into approximately 3‐mm pieces without mincing the tissue (Fig. 2A).

Figure 2.

Isolation of stromal cells from murine lung tissue. (A) Size of lung tissue pieces. (B) Example of the count setup after lung digestion. Viability is around 80% and live cell size is approximately between 4 and 20 µm.

• 4Carefully transfer the pieces into a C tube.
• 5Add 2.5 mL preheated digestion buffer to the tube containing the lung pieces.

  If digesting several lungs at once, place only one lung per tube.

  Digestion buffer needs to be preheated in the 37°C water bath before adding it to the lung tissue.

• 6Place the C tube in the dissociator. Ensure all tissue pieces are in the digestion medium and not sticking to the wall of the tube.
• 7Start the preset program called 37°C_m_LDK_1.

  This program is preinstalled and takes 31 min; it requires the dissociator to have a heating option as cells must be kept at 37°C during the entire isolation process to maintain enzymatic activity for cell dissociation.

• 8In the meantime, prepare collection tubes by placing precut filters on top of 15‐mL polypropylene centrifuge tubes. Prewet the filters with 1 mL precooled collection buffer.

  The collection buffer should be kept on ice for the entire duration of the preparation.

• 9After completion of the digestion program in the dissociator, briefly spin the C tube for 5 s to collect all material at the bottom of the tube.
• 10Filter the cell suspension through the prewetted 100‐µm filter into the 15‐mL polypropylene centrifuge tube.
• 11Wash the C tube with 2 mL precooled collection buffer and transfer over the 100‐µm filter to the 15‐mL polypropylene centrifuge tube.
• 12Wash the filter one more time with 1 mL precooled collection buffer to collect any cells sticking to the filter.
• 13Remove the filter and fill the 15‐mL polypropylene centrifuge tube completely with the precooled collection buffer.
• 14Centrifuge cells at 320 × g for 7 min at 4°C.
• 15Discard the supernatant and resuspend the pellet in 1 mL ACK lysis buffer per lung to lyse red blood cells.

  ACK lysis buffer should be kept at room temperature (RT).

• 16Incubate samples for 1 min at RT.

  It is crucial to not extend the incubation time, as fibroblasts become sensitive to lysis over time.

• 17Stop the lysis by adding ice‐cold PBS to the 15‐mL polypropylene centrifuge tube, filling it completely.

  This step dilutes and neutralizes the ACK lysis buffer.

• 18Centrifuge cells at 320 × g for 7 min at 4°C.
• 19Discard the supernatant and resuspend cells in 5 mL ice‐cold PBS per lung.

Count cells

• 20Take 10 µL of the cell suspension and add it to 10 µL trypan blue in an Eppendorf tube.
• 21Mix well and incubate at RT for 30 s.
• 22Fill 10 µL of this mixture into a cell counting chamber slide and load it onto the automated cell counter. Adjust the counting settings focusing on large cells, i.e., cells between 4 and 20 µm (Fig. 2B).

  Alternatively, manual cell counting could be performed here using the Neubauer chamber. However, that would result in higher inaccuracies in cell numbers due to the presence of cellular debris.

• 23The lung cell suspension is ready for Basic Protocols 2 and 3.

  We recommend processing a maximum of eight samples in parallel to ensure reproducible results, as this is all that can be accommodated at the same time on the dissociator. At the end of the isolation protocol, the yield should be around 30 × 10⁶ stromal cells per mouse lung. Immune cells are present in the cell suspension but with a much lower yield compared to that in typical immune isolation protocols used for the lung. The frequency of epithelial cells is lower than that in targeted cell isolation methods (Nakano et al., 2018). This inevitably accompanies the fibroblastic stromal enrichment due to the mechanical and enzymatic components of the protocol to disrupt the alveolar niche.

Basic Protocol 2. FLOW CYTOMETRY ASSESSMENT OF LUNG STROMAL POPULATIONS

This protocol aims to identify the fibroblastic stromal subsets including pericytes, alveolar fibroblasts, lipofibroblasts, adventitial fibroblasts, and peribronchial fibroblasts (Fig. 3 and Table 1). Additionally, their frequencies and surface marker expression (e.g., activation status) can be investigated this way using flow cytometry.

Figure 3.

Quantification and identification of recovered lung stromal cells. (A) FACS gating strategy for stromal cells in healthy mouse lung. ICAM expression of different stromal subsets is depicted. (B) Number of isolated cells per mouse lung. (C) Frequencies of PDGFRa⁺ fibroblasts, peribronchial fibroblasts, and pericytes shown as percentages of stromal cells.

Table 1.

Surface Marker Distribution for Lung Stromal Subsets

Stromal cell population (all CD45‐ Ter119‐)	Marker
Pericytes	Epcam‐ CD31‐ CD140a‐ CD146+ Sca1+ CD9‐/+
Adventitial fibroblasts	Epcam‐ CD31‐ CD140a+ CD146‐ Sca1+ CD9+
Alveolar fibroblasts	Epcam‐ CD31‐ CD140a+ CD146‐ Sca1‐ CD9‐
Lipofibroblasts	Epcam‐ CD31‐ CD140a+ CD146‐ Sca1‐ CD9‐ CD249+
Peribronchial fibroblasts	Epcam‐ CD31‐ CD140a‐ CD146‐ Sca1‐ CD9+
Lymphatic endothelial cells	Epcam‐ CD31+ gp38+
Blood endothelial cells	Epcam‐ CD31+ gp38‐
Type 1 epithelial cells	Epcam+ gp38+
Type 2 epithelial cells	Epcam+ gp38‐

Materials

• Lung cell suspension from Basic Protocol 1

• PBS, 1× (Thermo Fisher Scientific, cat. no. 10010023)

• eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific, cat. no. 65‐0865‐14)

• FC‐block mix (see recipe)

• Antibodies (see Table 2)

• FACS buffer (see recipe)

• Fixation solution (see recipe)

• FBS (Thermo Fisher Scientific, cat. no. 10082147)

• EDTA, 0.5 M (Thermo Fisher Scientific, cat. no. AM9260G)

• Sodium azide solution, 20% (Morphisto, cat. no. 18189.00100)

• BSA solution, 10% w/w (Miltenyi, cat. no. 130‐091‐376)

• FcR blocking reagent (Miltenyi, cat. no.130‐092‐575)

• Paraformaldehyde in PBS, 4% (Morphisto, cat. no. 10303.00500)

• Eppendorf tubes, 1.5 mL (Sarstedt, cat. no 72.706.400)

• Polypropylene centrifuge tubes, 15 mL (Greiner Bio‐One GmbH, cat. no. 188271)

• Table centrifuge (Eppendorf, cat. no. 5430 R)

• Filter mesh, 100 µm (cut into 1.5 × 1.5–cm cubes and autoclaved; A. Hartenstein GmbH, cat. no. PES3)

• BD LSR Fortessa II

Table 2.

Antibodies Used to Identify Lung Stromal Cell Populations

Antigen	Fluorochrome	Clone	Company	Catalog #	Stock conc. [mg/mL]	Dilution
CD45	Pe‐Cy7	30F11	BioLegend	103114	0.2	1:100
Ter119	Pe‐Cy7	Ter‐119	BioLegend	116222	0.2	1:100
CD31	AF488	MEC13.3	BioLegend	102514	0.5	1:200
Epcam	BV421	G8.8	BioLegend	118225	0.2	1:400
Gp38	APC	8.1.1	BioLegend	127410	0.2	1:100
CD140a	Pe	APA5	BioLegend	135906	0.2	1:100
CD146	BV711	ME‐9F1	BD Biosciences	740827	0.2	1:100
Sca‐1	PE‐Dazzle 594	D7	BioLegend	108137	0.2	1:600
CD9	PerCP‐Cy5.5	MZ3	BioLegend	124818	0.2	1:100
CD249	BV605	BP‐1	BD Biosciences	745238	0.2	1:2000

Staining cells for flow cytometry analyses

• 1Transfer 2 × 10⁶ cells from the cell suspension to a 1.5‐mL Eppendorf tube.
• 2Centrifuge cells at 320 × g for 5 min at 4°C.
• 3Resuspend the pellet in 500 µL PBS containing the viability dye at a dilution of 1:2000 and incubate for 30 min at 4°C in the dark.
• 4Add 500 µL PBS and centrifuge cells at 320 × g for 5 min at 4°C.
• 5Resuspend the cell pellet in 50 µL FC‐block mix and incubate for 5 min at 4°C in the dark.
• 6Add 50 µL antibody mix to each tube containing the 50 µL FC‐block mix. Mix with pipetting and incubate for 20 min at 4°C in the dark.
• 7Add 500 µL FACS buffer to each tube and centrifuge cells at 320 × g for 5 min at 4°C.
• 8Resuspend the cell pellet in 200 µL fixation solution and incubate for 10 min at RT in the dark.
• 9Add 500 µL PBS and centrifuge at 420 × g for 5 min at 4°C.
• 10Resuspend cell pellet in 250 µL FACS buffer and store at 4°C until measurement.

  Fixed samples can be stored for up to 2 weeks at 4°C.

• 11Filter samples over the 100‐µm precut filters before measurement.

  For setting up compensation, Ultra Comp eBeads Plus (Thermo Fisher Scientific, cat. no 01‐3333‐42) can be used following manufacturer instructions using the antibodies listed for the measurements (Table 2). It is advised to test the appropriate voltage for autofluorescence of the fibroblastic stromal populations using fluorescence minus one (FMO) controls. This helps set the right gating strategy as well (see Understanding Results).

• 12Measure samples on an LSR Fortessa II or a similar flow cytometer.

Basic Protocol 3. PURIFICATION OF LUNG FIBROBLASTIC STROMAL CELLS

This protocol describes how to purify fibroblastic stromal cells from lung single‐cell suspensions with high viability and purity for downstream functional application. The negative selection strategy results in untouched stromal cells that can be used for in‐vitro, scRNAseq, or lipidomics analyses (Fig. 4), whereas the alternative positive selection guide purifies alveolar and adventitial fibroblasts.

Figure 4.

Purification of lung fibroblastic stromal cells. (A) Main cell populations before and after purification of lung fibroblastic stromal cells. (B) Number of purified lung stromal cells per total lung.

Materials

• Lung single‐cell suspensions from Basic Protocol 2

• Dead cell removal kit (Miltenyi, cat. no. 130‐090‐101)

• Hanks balanced salt solution (HBSS) (Thermo Fisher Scientific, cat. no. 14025092)

• PBS, 1× (Thermo Fisher Scientific, cat. no. 10010023)

• MACS buffer (see recipe)

• CD45 MicroBeads, mouse (Miltenyi, cat. no. 130‐097‐153)

• CD31 MicroBeads, mouse (Miltenyi, cat. no. 130‐097‐418)

• Anti‐Ter‐119 MicroBeads, mouse (Miltenyi, cat. no. 130‐049‐901)

• CD326 (EpCAM) MicroBeads, mouse (Miltenyi, cat. no. 130‐105‐958)

• Polypropylene centrifuge tubes, 15 mL (Greiner Bio‐One GmbH, cat. no. 188271)

• Eppendorf tube, 1.5 mL (Sarstedt, cat. no 72.706.400)

• LS MACS column (Miltenyi, cat. no. 130‐042‐401)

• MACS manual magnetic separator including:

  • MidiMACS Separator (Miltenyi, cat. no. 130‐042‐302)

  • MACS MultiStand (Miltenyi, cat. no. 130‐042‐303)

• Overhead rotator (e.g., MACSmix Tube Rotator; Miltenyi, cat. no. 130‐090‐753)

• Centrifuge (Eppendorf, cat. no. 5810 R)

Dead cell removal

• 1Combine single‐cell suspensions from two lungs in one 15‐mL polypropylene centrifuge tube.
• 2Centrifuge the single‐cell suspensions at 320 × g for 7 min at 4°C.
• 3Remove the supernatant completely and resuspend the cell pellet in 100 µL dead cell removal microbeads per 10⁷ total cells.

  Dead cell removal microbeads are susceptible to bacterial contamination. Handle under sterile conditions. The minimal volume for separation is 500 µL. Add HBSS or binding buffer (included in the kit) to reach this volume. It is important to use HBSS with calcium and magnesium as the binding of dead cell removal microbeads (targeting Annexin V) is calcium dependent. The presence of the ion chelator EDTA will reduce binding.

• 4Mix well via careful pipetting and incubate for 15 min at RT.
• 5Prepare the LS column and place the column in the magnetic field of a suitable MACS manual magnetic separator.
• 6Place a precut 100‐µm filter on the column; rinse the column and prewet filter with 3 mL HBSS or Binding buffer (included in the kit).
• 7Apply the cell suspension onto the column via the filter and collect the flow‐through.
• 8Wash the column two times with 3 mL HBSS or Binding buffer and collect unlabeled cells that pass through and combine with the flow‐through from the previous step.
• 9Centrifuge flow‐through at 320 × g for 7 min at 4°C. Resuspend the pellet in 5 mL PBS.
• 10Count the cells as described in step 20 of Basic Protocol 1.

Negative selection of fibroblastic stromal cells

• 11Resuspend the cells in 90 µL MACS buffer per 10⁷ cells and transfer to a 1.5‐mL Eppendorf tube.
• 12Add CD45, CD31, Ter119, and Epcam microbeads (each 10 µL/ 10⁷ cells). Incubate the cell–microbead suspension for 15 min using a rotator at 4°C.

  We recommend this step be carried out in a 4°C fridge instead of incubating samples on ice.

• 13Transfer the cell–microbead suspension to a 15‐mL polypropylene centrifuge tube and fill the tube with MACS buffer.
• 14Centrifuge the cell–microbead suspension at 320 × g for 7 min at 4°C.
• 15Place the LS column on a MACS manual magnetic separator and rinse with 3 mL MACS buffer over a 100‐µm precut filter.
• 16Resuspend cells in MACS buffer (up to 10⁸ cells in 1 mL MACS buffer) and add to the column. Collect flow‐through.

  The LS column has maximal capacity for 1 × 10⁸ labeled cells and 2 × 10⁹ total cells. Due to the size of fibroblastic stromal cells, the maximal cell number should be avoided, and the total cells should be kept under 1.5 × 10⁹ cells.

• 17After applying the cells, wash the tube and filter with 600 µL MACS buffer; collect flow‐through and combine it with the that from the previous step.
• 18Wash the column three times with 3 mL MACS buffer; collect flow‐through and combine it with the that from the previous step.
• 19Centrifuge cells at 320 × g for 7 min at 4°C.
• 20Resuspend cells in 1 mL cell culture medium (for in‐vitro experiments) or PBS (for flow cytometry staining).

  If cells should be used in cell culture, include an additional washing step with the full medium to wash out any remaining EDTA from the MACS buffer. EDTA will decrease cell attachment in the culture flask.

• 21Count cells as described in step 20 of Basic Protocol 1.
• 22Take an aliquot of cells (5 × 10⁴ cells) to determine the purity of isolation via flow cytometry. Follow the staining procedure described in Basic Protocol 2. The antibodies to use and their concentrations are listed in Table 3.

  It is necessary to use FMO controls to determine gating for purity check.

  This strategy will purify a bulk population of untouched fibroblastic stromal cells. Alternatively, PDGFRa⁺ fibroblasts, including alveolar and adventitial fibroblasts, can be positively selected over PDGFRa^‐ stromal cells. Follow alternate protocol, starting after step 10 of Basic Protocol 3 (Dead cell removal).

Table 3.

Antibodies Used to Validate Purity of Lung Stromal Cell Isolation

Antigen	Fluorochrome	Clone	Company	Catalog #	Stock conc. [mg/ml]	Dilution
CD45	APC‐Cy7	30F11	BioLegend	103116	0.2	1:100
Ter119	Pe‐Cy7	Ter‐119	BioLegend	116222	0.2	1:100
CD31	AF488	MEC13.3	BioLegend	102514	0.2	1:200
Epcam	BV421	G8.8	BioLegend	118225	0.2	1:400
Gp38	APC	8.1.1	BioLegend	127410	0.2	1:100

POSITIVE SELECTION OF FIBROBLASTIC STROMAL CELLS

Additional materials

• FcR Blocking Reagent, mouse (Miltenyi, cat. no.130‐092‐575)

• Biotin anti‐mouse CD140a Antibody (BioLegend, cat. no. 135910)

• Anti‐Biotin Microbeads (Miltenyi, cat. no. 130‐090‐485)

• 1Resuspend the cells in 90 µL MACS buffer (see recipe) per 10⁷ cells and transfer them to a 1.5‐mL Eppendorf tube.
• 2Add 10 µL FcR blocking reagent per 90 µL MACS buffer.
• 3Incubate cells for 5 min at 4°C.
• 4Add 6 µL anti‐mouse CD140a antibody per10⁷ cells.
• 5Incubate cell suspension for 10 min at 4°C.

  We recommend this step be carried out in a 4°C fridge instead of incubating samples on ice.

• 6Transfer the cell suspension to a 15‐mL polypropylene centrifuge tube and fill up the tube with MACS buffer.
• 7Centrifuge the cell suspension at 320 × g for 7 min at 4°C.
• 8Resuspend in 80 µL MACS buffer, add 20 µL anti‐biotin microbeads per every 10⁷ cells, and mix with gentle pipetting.
• 9Incubate the cell–microbead suspension for 15 min in a rotator at 4°C.

  We recommend this step be carried out in a 4°C fridge instead of incubating samples on ice.

• 10Transfer the cell–microbead suspension to a 15‐mL polypropylene centrifuge tube and fill up the tube with MACS buffer.
• 11Centrifuge the cell–microbead suspension at 320 × g for 10 min at 4°C.
• 12Place the LS column on the MACS manual magnetic separator and rinse with 3 mL MACS buffer over a 100‐µm precut filter.
• 13Resuspend cells in MACS buffer (up to 10⁸ cells in 1 mL MACS buffer) and apply to the column.

  The LS column has maximal capacity for 1 × 10⁸ labeled cells and 2 × 10⁹ total cells. Due to the size of fibroblastic stromal cells, the maximal cell number should be avoided, and the total cells should be kept under 1.5 × 10⁹ cells.

• 14After applying the cells, wash the tube and filter with 600 µL MACS buffer.
• 15Wash the column three times with 3 mL MACS buffer and discard flow‐through (contains CD140a^‐ fraction).
• 16Remove the column from the separator and place it on top of a 15‐mL tube.
• 17Add 5 mL of MACS buffer to the column. Collect flow‐through (contains CD140a⁺ fraction).

  We do not recommend using a plunger as this will result in decreased cell viability.

• 18Centrifuge cells at 320 × g for 7 min at 4°C.
• 19Resuspend cells in 1 mL cell culture medium (for in‐vitro experiments) or PBS (for flow cytometry staining).

  If cells should be used in cell culture, include an additional washing step with full medium to wash out any remaining EDTA from the MACS buffer. EDTA will decrease cell attachment in the culture flask.

• 20Count cells as described in Basic Protocol 1, step 20.

REAGENTS AND SOLUTIONS

Collection buffer

• PBS

• 0.5% FBS

• 10 mM EDTA

Digestion buffer

• RPMI medium

• 2% FBS

• 20 mM HEPES

• 1 mg/mL Collagenase P

• 1 mg/mL Collagenase II

• 25 µg/mL DNase I

FACS buffer

• PBS

• 0.1% sodium azide

• 0.4% (v/v) BSA

• 2 mM EDTA

FC‐block mix

• 10 µL FC‐block in 40 µL FACS buffer per staining

Fixation solution

• PBS

• 2% paraformaldehyde

MACS buffer

• PBS

• 2% FBS

• 2 mM EDTA

COMMENTARY

Background Information

We developed this isolation and purification strategy given the growing interest in the understanding of the lung fibroblastic stromal compartment. The central advantage of this isolation method is that it ensures high yield and viability of the stromal compartment, specifically the fibroblastic stromal cells. The specific surface marker panel allows for a deeper understanding of these cells using flow cytometry readouts e.g., activation state, surface molecule expression, or lipid uptake. The purification method is fast and typically produces over 95% pure fibroblastic stromal cells. Although the stromal cells represent a bulk population after this purification technique, they can be used for in‐vitro expansion, various omics applications, or further functional assays.

Critical Parameters

Cell isolation: viability

Shear stress, a force generated by fluid pressure on the cell surface, is detected and transduced into biochemical signals by the cell (Espina et al., 2023). Such mechanotransduction changes the transcriptional profile and migratory behavior, and most importantly affects cell viability (Garanich et al., 2007; Grierson & Meldolesi, 1995; Mourgeon et al., 1999). During isolation procedures, mechanical disruption and fluid shear stress generated from pipetting are unavoidable. Large cells, such as fibroblasts, are especially sensitive and can respond with reduced viability upon stringent mechanical perturbation. Therefore, it is critical to avoid extensive mixing using pipette tips with smaller diameters. We recommend mixing stromal cell suspensions using 1‐mL pipet tips with larger‐diameter orifices.

To increase the isolated cell number, pooling of several mice will likely be necessary, However, we do not recommend purification with the autoMACS separator under these circumstances as cell viability will be reduced considerably. We suggest distributing the samples into several LS columns and performing cell separation manually, rather than with an automated system.

Due to the embedding of stromal cells into the extracellular matrix, the use of enzymes is necessary for the isolation. Nonetheless, cells must be processed without interruptions and not exposed for an extended time to the digestion buffer.

Cell purity after negative selection

Typically, cell purity is over 95% with a yield of 6 × 10⁵ fibroblastic stromal cells per lung when following this protocol. The good yield and purity ensure that cells are suitable for downstream functional analyses. The removal of dead cells is key to avoiding the nonspecific binding of dead cells to the microbeads, thereby reducing cell yield and purity. We recommend maintaining the total cell number to beads ratio for dead cell removal and negative selection. It is therefore essential to count samples as suggested to ensure reproducible results. Incorrect high cell numbers will not only reduce yield and purity but can also completely clog the LS column due to the large size of fibroblasts.

Troubleshooting

Common problems, their causes, and possible solutions are listed in Table 4.

Table 4.

Troubleshooting

Problem	Possible cause	Solution
Tissue pieces left after lung digestion	Tissue pieces were sticking to the wall of the tube and were not immersed completely in the digestion buffer. Enzymes are not properly aliquoted, and activity is low. Digestion buffer is not prewarmed.	Ensure all tissue pieces are in the digestion buffer. Always use HBSS for collagenase stock preparation. Always prewarm digestion buffer. The gentleMACS heating panel and digestion time are not enough to warm the solution; make sure tissue digestion occurs within the 31‐min run time.
DNA gel formation after lung digestion/ACK lysis	No DNase is added to digestion buffer.	Always prepare the digestion buffer freshly with the enzymes. Filter cell suspension after ACK lysis over a 100‐µm filter. Pipette cell suspension more carefully.
Very low cell viability after MACS purification (<90%)	Harsh pipetting was done. Cells were pressed through the column with a plunger. A very high cell number was used for isolation (inaccurate cell counting).	Avoid pipetting against the bottom of the 15‐mL polypropylene centrifuge tube to decrease cell mechanical stress. Avoid mixing cell suspensions with pipettes other than 1‐mL pipette tips. Double‐check cell counting. Cell viability can be increased by enriching live cells over a second LS column.
Low cell purity	Cell counting is inaccurate.	Ensure the correct total cell number to beads ratio is kept. Make sure cells are resuspended properly before the addition of microbeads.

Statistical and Data Analysis

Data are presented as mean ± standard error of the mean. Figures were generated using Prism 9 (GraphPad Software Inc., La Jolla, California, USA) or using BioRender.com. Flow cytometry data were analyzed using FlowJo 10.8.1 (FlowJo LLC, Ashland, Oregon, USA).

Understanding Results

The primary aim of this protocol is to isolate lung stromal cells, specifically the fibroblastic stromal compartment. The cells can be analyzed comprehensively for different fibroblast subsets. Figure 3 depicts a representative result of the lung stromal cell isolation together with a detailed gating strategy. After gating out dead cells, doublets, CD45⁺ (immune cells), Ter119⁺ (remaining red blood cells), and stromal cells are defined by PDGFRa and CD146 expression as either pericytes (CD146⁺ and PDGFRa^‐) or fibroblasts (CD146^‐ and PDGFRa^‐/+). Fibroblasts can be further defined as PDGFRa⁺ Sca1⁺ CD9⁺ adventitial fibroblasts, PDGFRa^‐ CD9⁺ peribronchial fibroblasts, and PDGFRa⁺ Sca1^‐ alveolar fibroblasts. CD249 is highly expressed on alveolar fibroblasts and can be used to specifically distinguish lipofibroblasts, a lipid‐storing fibroblast population localized within the alveolar niche (Fig. 3A).

We plotted the fibroblastic activation marker ICAM1 expressions as examples for further flow cytometry exploration of the cells after following the above‐described isolation protocol (Fig. 3A). Notably, endothelial cells and type 1 and type 2 epithelial cells can be also distinguished with this isolation protocol. The surface marker expressions are given in Table 1. Finally, the typical frequency distribution of the isolated subsets is depicted in Figure 3C, and the purity check after negative selection is in Figure 4.

Time Considerations

The preparation of reagents usually requires 20‐30 min. The isolation of cells takes approximately 1 h, including the time needed for automated digestion. When processing multiple samples in parallel, the isolation time can be extended up to 1.5 h. Further, cell purification takes an additional 2 h while the flow cytometry staining requires 1.5 h. If the cells are used for in‐vitro assays, we recommend to plate cells without delay.

Author Contributions

Sophia Rottmann: Data curation; formal analysis; methodology; writing—original draft. Veronika Lukacs‐Kornek: Conceptualization; funding acquisition; supervision; writing—original draft; writing—review and editing.

Conflict of Interest

There are no conflicts of interest.

Rottmann, S. , & Lukacs‐Kornek, V. (2024). Isolation, purification, and comprehensive flow cytometry assessment of lung stromal cells. Current Protocols, 4, e70078. doi: 10.1002/cpz1.70078

Data Availability Statement

The data that support the protocol are available from the corresponding author upon reasonable request.