Protocol for the isolation, enrichment, and culture of murine adipose progenitor cells

Summary

We present a protocol for isolating and culturing murine adipose progenitor cells (APCs) from adipose tissue. We describe steps for tissue dissection, unit-normalized enzymatic digestion, stromal vascular fraction (SVF) isolation, APC enrichment, and cell culture. We then detail quality control procedures that maximize cell yield, viability, and purity. This protocol combines enzymatic SVF isolation with magnetic-activated cell sorting enrichment to obtain a highly enriched APC population. It emphasizes reproducibility through unit-normalized digestion, controlled processing conditions, and standardized quality control metrics.

Graphical abstract

Highlights

• •Isolate stromal vascular fraction (SVF) from murine adipose tissue
• •Enrich adipose progenitor cells (APCs) using MACS
• •Culture and expand murine APCs under standardized conditions
• •Assess SVF/APC yield, viability, and purity for quality control

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

We present a protocol for isolating and culturing murine adipose progenitor cells (APCs) from adipose tissue. We describe steps for tissue dissection, unit-normalized enzymatic digestion, stromal vascular fraction (SVF) isolation, APC enrichment, and cell culture. We then detail quality control procedures that maximize cell yield, viability, and purity. This protocol combines enzymatic SVF isolation with magnetic-activated cell sorting enrichment to obtain a highly enriched APC population. It emphasizes reproducibility through unit-normalized digestion, controlled processing conditions, and standardized quality control metrics.

Before you begin

The protocol below describes the isolation and culture of adipose progenitor cells (APCs) from murine inguinal white adipose tissue (iWAT). The workflow can also be applied to visceral white adipose tissue (vWAT) and brown adipose tissue (BAT) with optimization of tissue digestion.

• 1.Maintain all samples, buffers, and reagents at 2°C–8°C except during enzymatic digestion.
• 2.Perform all centrifugation steps at 4°C unless otherwise specified.
• 3.Process adipose tissue immediately after collection to preserve cell viability.
• 4.Define and record the enzyme type, enzyme activity (U/mL), and digestion time before beginning the protocol.

CRITICAL: Standardize tissue washing, red blood cell removal, digestion conditions, and centrifugation parameters across experiments to minimize technical variability.

CRITICAL: Normalize digestion volume, MACS labeling reagents, and cell seeding density to the initial tissue mass (g) to ensure reproducibility between experiments.

Innovation

This protocol provides a standardized workflow for isolating, enriching, and culturing murine adipose progenitor cells (APCs) from the stromal vascular fraction (SVF).¹ We use unit-normalized enzymatic digestion (U/mL), rather than conventional mass-based reporting, to promote consistent tissue dissociation across enzyme lots.² We also standardize critical pre-analytical variables, including tissue handling, temperature control, washing, filtration, centrifugation, and red blood cell removal, which are often incompletely reported.³ In addition, we incorporate magnetic-activated cell sorting (MACS) for lineage depletion and APC enrichment into a single reproducible workflow while preserving cell viability. By standardizing interdependent processing parameters rather than individual steps, this protocol improves transparency, reproducibility, and cross-study comparability in studies of APC biology.

Institutional permissions

The Animal Care Committee of McMaster University and Hamilton Health Sciences approved all animal procedures in accordance with the Canadian Council on Animal Care (CCAC) guidelines (McMaster University Animal Research Ethics Board [AREB], Hamilton, Ontario, Canada; AUP# 22-08-30). We maintained mice on a C57BL/6J background in a specific pathogen-free facility under controlled environmental conditions (12-h light/dark cycle, ∼22°C, ∼50% humidity) and provided food and water ad libitum. We used male mice (10–12 weeks old) for stromal vascular fraction (SVF) isolation.

Preparation 1: Prepare complete culture medium

Timing: 15 min

• 5.Prepare complete culture medium by supplementing DMEM/F-12 with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin-streptomycin.
• 6.Mix thoroughly by gentle inversion.
• 7.Warm only the required volume to 37°C immediately before use.

CRITICAL: Avoid repeated warming and cooling of complete medium, as this may reduce the stability of serum components and antibiotics.

Pause point: Store complete medium at 2°C–8°C for up to 4 weeks according to the manufacturer's recommendations.

Preparation 2: Prepare digestion medium

Timing: 20 min

• 8.Prepare fresh digestion medium immediately before tissue processing according to the recipe provided in step 3 of the “step-by-step method details” section.

CRITICAL: Prepare digestion medium immediately before use because proteolytic enzyme activity decreases during storage.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-CD45, APC (diluted 1:100)	Thermo Fisher Scientific	Cat# 17-0451-82
Anti-CD31, APC (diluted 1:100)	Thermo Fisher Scientific	Cat# 17-0311-82
Anti-Ter119, APC (diluted 1:100)	Thermo Fisher Scientific	Cat# 17-5921-82
LIVE/DEAD Fixable Dead Cell Stain Kit (diluted 1:1000)	Thermo Fisher Scientific	Cat# L34957
Anti-Sca-1, Pacific Blue (diluted 1:100)	BioLegend	Cat# 108120
Anti-PDGFRα, PE (diluted 1:100)	BioLegend	Cat# 135905
Chemicals, peptides, and recombinant proteins
Collagenase D	Roche	Cat# 11088882001
Dispase II	Roche	Cat# 04942078001
CaCl2	Thermo Fisher Scientific	Cat# L13191.30
Trypan Blue Solution	Gibco	Cat# 15250061
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	Cat# D4540
Dulbecco’s PBS (DPBS), without Ca2+ and Mg2+	Wisent	Cat# 311-425-CL
Critical commercial assays
Adipose Progenitor Cell Isolation Kit (mouse)	Miltenyi Biotec	Cat# 130-106-639
Experimental models: Organisms/strains
Mouse: Wild-type, C57BL/6J, male, 10–12 weeks old	The Jackson Laboratory	Stock No. 000664
Software and algorithms
FlowJo v11	BD Biosciences	https://www.flowjo.com
GraphPad Prism v10	GraphPad Software	https://www.graphpad.com
Other
DMEM/F-12 medium	Gibco	Cat# 12634010
Fetal bovine serum (FBS)	Thermo Fisher Scientific	Cat# 10082147
ACK Lysing Buffer	Thermo Fisher Scientific	Cat# J62150.AP
L-glutamine	Thermo Fisher Scientific	Cat# 25030081
Penicillin–streptomycin	Wisent	Cat# 450-201-ZL
MACS Rinsing Buffer	Miltenyi Biotec	Cat# 130-091-222
MACS MultiStand	Miltenyi Biotec	Cat# 130-042-303
LS Columns	Miltenyi Biotec	Cat# 130-042-401
MS Columns	Miltenyi Biotec	Cat# 130-042-201
40 μm Cell Strainer	Sarstedt	Cat# 83.1826.001
24-well Cell Culture Plate	Corning	Cat# 3524

Storage: Prepare fresh immediately before use. Keep on ice until use. Do not store after enzyme addition.

CRITICAL: Collagenase D and Dispase II are proteolytic enzymes that may cause skin, eye, and respiratory irritation. Wear appropriate personal protective equipment (lab coat, gloves, and eye protection), avoid aerosol generation, and prepare the digestion medium in accordance with institutional laboratory safety guidelines.

Alternatives: Collagenase type I or type II may be substituted for Collagenase D after optimization of enzyme activity and digestion time. Other neutral proteases (e.g., Liberase blends or Dispase I) may also be suitable but require protocol optimization because enzyme composition and activity differ between preparations.

Step-by-step method details

Dissection of AT from mice

Timing: 30 min

This step describes the isolation of adipose tissue depots from mice for downstream applications. Rapid and sterile tissue collection is essential to preserve tissue integrity, cellular viability, and molecular profiles.

• 1.
  Begin adipose tissue collection immediately following euthanasia.

  • a.
    Spray the mouse fur thoroughly with 70% ethanol before dissection to minimize contamination.
  • b.
    Immediately transfer each excised adipose tissue depot to a 6-well culture plate containing sterile, ice-cold DPBS to preserve tissue integrity and cell viability.
• 2.
  Dissect the required adipose tissue depot.

  • a.
    Cut through the skin without disrupting the peritoneum.
  • b.
    Stretch the skin laterally and secure it with needles to expose the underlying tissue.
  • c.
    For iWAT, identify the bilateral fat depots beneath the skin extending from the lower back along the inner thighs toward the inguinal region (Figure 1A).
  • d.
    For vWAT, access the peritoneal cavity and carefully isolate the visceral fat pads while avoiding contamination from surrounding organs (Figure 1B).
  • e.
    For BAT, locate the darker, lobular depot beneath the dorsal skin between the shoulder blades and gently separate it from the adjacent white adipose tissue (Figure 1C).

Note: Although this protocol applies to SVF isolated from iWAT, vWAT, and BAT, we used iWAT-derived SVF for protocol optimization and the representative flow cytometric validation shown in Figure 4.

CRITICAL: Work quickly and carefully during dissection to avoid contamination from surrounding tissues. Immediately place excised adipose tissue in sterile, cold DPBS to preserve cell viability and minimize degradation before downstream processing.

Figure 1.

Adipose tissue depots collected for SVF isolation

Representative images showing the anatomical locations of adipose tissue depots harvested from mice for downstream SVF isolation and APC analysis. Red boxes indicate the regions of adipose tissue collected from each depot.

(A) Bilateral inguinal white adipose tissue (iWAT) depots.

(B) Visceral white adipose tissue (vWAT) depots.

(C) Interscapular brown adipose tissue (BAT) depot. Tissues were carefully dissected prior to enzymatic digestion and SVF isolation.

Figure 4.

Flow cytometric validation of MACS-enriched adipose progenitor cells (APCs) from murine iWAT

(A) Representative sequential gating strategy used for APC identification. Cells were first identified based on FSC-A and SSC-A, followed by singlet discrimination using FSC-A and FSC-H. Viable cells were identified as LIVE/DEAD™ Fixable Aqua-negative cells. Lineage-positive cells were subsequently excluded based on CD45, CD31, and Ter119 expression to identify the live CD45⁻CD31⁻Ter119⁻ population.

(B) Representative Sca-1 versus PDGFRα flow-cytometry plots before and after MACS enrichment. APCs were defined as lineage-negative Sca-1⁺PDGFRα⁺ double-positive cells. The upper-right quadrant (Q2) represents the Sca-1⁺PDGFRα⁺ APC population.

(C) Quantification of Sca-1⁺PDGFRα⁺ APCs in the flow-through and APC-enriched fractions, expressed as a percentage of total live CD45⁻CD31⁻Ter119⁻ cells. Data are presented as mean ± SEM. ∗∗∗∗p < 0.0001.

Enzymatic digestion to isolate SVF

Timing: 2 h

This step describes the enzymatic dissociation of AT to isolate the SVF, a heterogeneous cell population containing APCs, immune cells, endothelial cells, pericytes, and other stromal populations. Efficient tissue digestion and careful removal of mature adipocytes and erythrocytes are critical for maximizing cell yield, viability, and reproducibility in downstream applications such as flow cytometry, cell sorting, cell culture, gene expression analysis, and functional assays.

• 3.Prepare fresh digestion medium at a volume of 1 mL per gram of tissue, containing Collagenase D at 1.5 U/mL, Dispase II at 2.4 U/mL, and CaCl₂ at 10 mM.

CRITICAL: Record enzyme lot numbers and activity (U/mL). Reporting enzyme concentration in mg/mL alone is discouraged due to lot-to-lot variability.

• 4.
  Prepare the adipose tissue for enzymatic digestion.

  • a.
    Dissolve the enzymes in the required volume of DPBS.
  • b.
    Mix thoroughly and keep the digestion medium on ice until use.
  • c.
    Weigh the adipose tissue in a sterile environment.
  • d.
    Transfer the tissue to an appropriate digestion tube.
  • e.
    Add digestion medium at 1 mL per gram of tissue.

CRITICAL: Carefully remove non-adipose contaminants (e.g., connective tissue and hair), as these can alter downstream stromal and immune cell composition.

• 5.Finely mince AT using sterile scissors to generate small, uniform fragments, facilitating efficient enzymatic digestion (Figure 2A).
• 6.
  Incubate the samples at 37°C with continuous rotation at 20–30 rpm.

  • a.
    Gently vortex or shake the tubes every 10 min to ensure uniform enzymatic exposure.
  • b.
    Digest BAT for 30–35 min, iWAT for 35–40 min, or vWAT for 40–50 min using the enzyme concentrations specified in this protocol.
  • c.
    Monitor tissue dissociation throughout the incubation and stop digestion once the tissue is fully dispersed to avoid overdigestion and preserve cell viability.

CRITICAL: Digestion time is depot-dependent. Avoid extending digestion beyond the depot-specific range stated above, and stop once the tissue is sufficiently dissociated.

• 7.
  Terminate enzymatic digestion.

  • a.
    Add complete medium containing DMEM/F-12 supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin–streptomycin.
  • b.
    Mix thoroughly by gentle pipetting until the suspension appears homogeneous (Figure 2B).
• 8.Centrifuge the suspension at 500 × g for 10 min at 20°C–25°C.

Note: Following centrifugation, a floating adipocyte/lipid layer and the SVF pellet should be visible (Figure 2C).

• 9.Carefully aspirate and discard the floating lipid and adipocyte layers without disturbing the SVF pellet.
• 10.Resuspend the SVF pellet in 1 mL of ACK red blood cell lysis buffer and incubate on ice for 4 min.
• 11.Gently invert tubes every 1 min during RBC lysis to ensure uniform exposure.

CRITICAL: Limit RBC lysis to 4 min, as prolonged exposure may reduce cell viability.

• 12.
  Neutralize the RBC lysis reaction immediately after 4 min.

  • a.
    Add 2 mL of complete medium.
  • b.
    Centrifuge the cells at 500 × g for 10 min.

Note: Although RBC lysis efficiently removes mature erythrocytes, residual nucleated erythroid-lineage cells may remain within the SVF. Therefore, Ter119 is included during downstream flow cytometric analysis to identify and exclude residual erythroid-lineage cells when assessing APC purity.

• 13.
  Wash the SVF pellet.

  • a.
    Carefully aspirate and discard the supernatant.
  • b.
    Resuspend the cell pellet in 10 mL of complete medium.
  • c.
    Centrifuge at 500 × g for 10 min (Figure 2D).
• 14.
  Filter the cell suspension.

  • a.
    Pre-wet a 40-μm cell strainer with complete medium.
  • b.
    Pass the cell suspension through the strainer into a 50-mL tube.
  • c.
    Centrifuge at 500 × g for 10 min and retain the SVF pellet.

CRITICAL: Pre-wet the strainer with medium to minimize cell loss.

• 15.Use the resulting SVF pellet immediately for cell counting.

Figure 2.

Representative workflow of SVF isolation from adipose tissue

(A) Minced adipose tissue suspended in digestion medium prior to enzymatic dissociation.

(B) Adipose tissue following enzymatic digestion, showing progressive tissue dissociation and liberation of stromal vascular components.

(C) Formation of the SVF-containing pellet following centrifugation of digested adipose tissue.

(D) SVF suspension following erythrocyte lysis and washing, demonstrating removal of contaminating erythrocytes and preparation of the SVF for downstream flow cytometry, MACS enrichment, cell culture, and molecular analyses.

Cell counting and viability assessment

Timing: 30 min

This step determines the total SVF cell yield and viability following tissue digestion and isolation. Accurate cell counting is essential for standardizing downstream applications. Assessing viability also provides an important quality control measure, allowing researchers to evaluate the efficiency of tissue processing and identify potential issues arising from over-digestion, prolonged handling, or cell loss during isolation.

• 16.Resuspend SVF pellet in 1 mL complete medium.

Note: To assess cell viability, mix a 10 μL aliquot of the cell suspension with an equal volume of 0.4% trypan blue (1:1, v/v). Count viable, trypan blue-negative cells using a hemocytometer or automated cell counter.

Note: For SVF preparations intended for downstream culture or cell sorting, a minimum viability of ≥70% is generally acceptable. For optimal culture initiation and experimental consistency, a viability of ≥80% is recommended.

• 17.
  Wash the cells with cold MACS rinsing buffer.

  • a.
    Centrifuge the cell suspension at 500 × g for 10 min.
  • b.
    Carefully aspirate and discard the supernatant.
  • c.
    Resuspend the cell pellet in 10 mL of cold MACS rinsing buffer.
  • d.
    Centrifuge again at 500 × g for 10 min.
  • e.
    Discard the supernatant and retain the cell pellet.

MACS enrichment and depletion for APC isolation

Timing: 1 h

This step enriches adipose progenitor cells (APCs) from the stromal vascular fraction (SVF) by depleting lineage-positive non-adipocyte cells using the Adipose Progenitor Cell Isolation Kit (Miltenyi Biotec), which contains a proprietary depletion cocktail. APC enrichment is verified by downstream flow cytometry using lineage exclusion markers (CD45, CD31, and Ter119) together with APC markers (Figure 4).

• 18.Resuspend the cell pellet in 80 μL of pre-cooled MACS rinsing buffer per gram of starting tissue, as recommended by the manufacturer.

Note: If the recovered cell number has been determined prior to magnetic labeling, adjust the MACS rinsing buffer volume to maintain a maximum concentration of 1 × 10⁷ cells per 80 μL of MACS rinsing buffer. For example, use 160 μL of MACS rinsing buffer for up to 2 × 10⁷ cells or 240 μL for up to 3 × 10⁷ cells to ensure optimal labeling efficiency.

• 19.
  Label the cells with the Non-Adipocyte Progenitor Depletion Cocktail.

  • a.
    Add 20 μL of Non-Adipocyte Progenitor Depletion Cocktail per 1 × 10⁷ cells.
  • b.
    Adjust the labeling volume based on the total cell number; use tissue-normalized scaling as an initial approximation when required.
  • c.
    Mix gently and thoroughly.
  • d.
    Fully resuspend the cell pellet and minimize clumping to permit unobstructed flow through the column.

Note: The exact antibody composition of the commercial Non-Adipocyte Progenitor Depletion Cocktail is proprietary and is not disclosed by the manufacturer. In this protocol, successful depletion is confirmed by downstream flow cytometry demonstrating enrichment of lineage-negative (CD45⁻CD31⁻Ter119⁻) cells prior to APC characterization.

• 20.Incubate in the dark for 15 min at 2°C–8°C.

CRITICAL: Avoid prolonged incubation or higher temperatures, as these may increase non-specific labeling.

CRITICAL: Do not exceed manufacturer-recommended cell capacity (typically ≤10⁷ total cells per LS column).

• 21.Place the LS MACS column into the magnetic separator and position a sterile collection tube beneath it.
• 22.Rinse the column with degassed 3 mL MACS rinsing buffer.
• 23.
  Apply the labeled cell suspension to the equilibrated column.

  • a.
    Allow the cell suspension to pass through the column by gravity.
  • b.
    Collect the flow-through containing unlabeled cells (Figure 3A).

CRITICAL: Allow gravity flow. Add subsequent volumes only when the column reservoir is empty.

• 24.Wash the column with 3 × 500 μL MACS rinsing buffer to remove residual unlabeled cells.

Note: To increase purity, reapply the collected fraction to a new column. This additional separation step may reduce overall cell yield.

Figure 3.

Representative workflow of magnetic-activated cell sorting (MACS)-based cell enrichment

(A) Negative selection using an LS column. Magnetically labeled non-APC populations are retained in the column, whereas the unlabeled, APC-containing fraction passes through and is collected.

(B) Positive selection using an MS column. Magnetically labeled APCs are retained in the column and subsequently eluted after removal of the column from the magnetic separator.

Positive selection of APCs

Timing: 1 h

This step further enriches the APC population through positive magnetic selection following initial depletion of non-APC populations. Positive selection increases APC purity by isolating cells expressing APC-specific surface markers, generating a highly enriched population suitable for downstream applications requiring maximal purity, including cell culture, differentiation assays, flow cytometry, gene expression analysis, metabolic studies, and functional characterization.

The Adipocyte Progenitor Isolation Cocktail is a proprietary reagent supplied as part of the commercial kit. The manufacturer does not disclose the specific surface marker(s) targeted by the magnetic beads. Consequently, we verified APC enrichment by downstream flow cytometry using lineage exclusion (CD45, CD31, Ter119) together with Sca-1 and PDGFRα expression.

• 25.
  Pellet the pre-enriched cell suspension.

  • a.
    Centrifuge the cell suspension at 500 × g for 10 min.
  • b.
    Carefully aspirate and discard the supernatant.
• 26.
  Label the cells with the Adipocyte Progenitor Isolation Cocktail.

  • a.
    Add 80 μL of MACS rinsing buffer and 20 μL of Adipocyte Progenitor Isolation Cocktail per 1 × 10⁷ cells.
  • b.
    Scale both volumes proportionally according to the total cell number.

Note: For example, use 160 μL of MACS rinsing buffer and 40 μL of Adipocyte Progenitor Isolation Cocktail for 2 × 10⁷ cells.

• 27.Mix well and incubate for 15 min in the dark in the refrigerator (2°C–8°C).
• 28.
  Prepare the MS column for positive selection.

  • a.
    Place a fresh MS column in the magnetic separator.
  • b.
    Equilibrate the column with 500 μL of MACS rinsing buffer.

Note: Use an MS column for the positive-selection step, as recommended by the manufacturer for isolating adipocyte progenitor cells with the Adipocyte Progenitor Isolation Kit.

Note: The MS Column is optimized for the expected number of positively labeled progenitor cells, whereas the LS Column is used during the lineage depletion (negative-selection) step because it accommodates the larger number of magnetically labeled lineage-positive cells. Use the same column type consistently throughout each respective separation step.

• 29.Apply the pre-enriched cell suspension onto the column and collect the unlabeled fraction (Figure 3B).
• 30.Wash the column with 3 × 500 μL of MACS rinsing buffer to remove residual unlabeled cells. Retain the flow-through separately if required for downstream analysis.
• 31.
  Elute the APC-enriched fraction.

  • a.
    Remove the column from the magnetic separator.
  • b.
    Place the column over a new collection tube.
  • c.
    Add 1 mL of MACS rinsing buffer.
  • d.
    Immediately flush the column using the plunger to collect the magnetically labeled APC-enriched fraction.
• 32.Count the cells as described above to determine the appropriate cell number for subsequent cell culture experiments.

Note: Representative Sca-1 versus PDGFRα flow cytometry analysis before and after MACS enrichment (Figure 4B), together with quantification of lineage-negative Sca-1⁺PDGFRα⁺ APCs as a percentage of live CD45⁻CD31⁻Ter119⁻ cells (Figure 4C), demonstrates successful APC enrichment.

• 33.If the cells are intended for culture, wash them once with complete culture medium containing DMEM/F-12, 10% FBS, 1% L-glutamine, and 1% penicillin–streptomycin to remove residual EDTA and proteins and standardize downstream conditions.

Culture conditions for APCs

Timing: 60 min

This step establishes standardized culture and cryopreservation conditions for isolated APCs, with appropriate surface preparation, seeding density, medium maintenance, and passaging practices supporting cell attachment, viability, proliferation, and preservation of progenitor characteristics. Standardized culture conditions improve reproducibility across experiments and minimize variability in downstream functional, differentiation, and molecular analyses.

• 34.Dilute the cell suspension based on the cell count to achieve the desired seeding density.
• 35.Seed 5 × 10⁴ cells per well in a 24-well plate.

Note: Under these conditions, cells typically reach approximately 80%–90% confluence within 24–36 h.

CRITICAL: Define and report standardized seeding densities for both initial plating (P0) and post-passaging conditions.

Note: Adjust the seeding density according to experimental requirements. Lower densities may delay attachment, whereas higher densities may accelerate confluence and alter cell behavior.

• 36.
  Maintain cultures at 37°C and 5% CO₂ in a humidified incubator.

  • a.
    Replace the medium approximately 4 h after seeding to remove non-adherent cells.
  • b.
    Replace the medium every 2–3 days thereafter.
  • c.
    Passage cells at approximately 80%–90% confluence.

CRITICAL: Record all passaging parameters, including dissociation reagent, incubation time, neutralization method, centrifugation conditions, and reseeding density, to ensure reproducibility.

Note: Routinely test cells for mycoplasma contamination before downstream applications.

Note: Representative passage-0 (P0) APC cultures at approximately 80%–90% confluence are shown in Figure 5A. At this stage, cells display the characteristic fibroblast-like morphology of cultured APCs. Flow cytometric assessment of Sca-1 expression at confluence demonstrates maintenance of a predominantly Sca-1-positive population (Figure 5B).

• 37.
  Freeze cells at 80%–90% confluence to maximize post-thaw recovery.

  • a.
    Prepare cryopreservation medium containing 80% FBS, 10% DMSO, and 10% DMEM/Ham’s F-12.
  • b.
    Prepare the cryopreservation medium fresh or store it for no longer than 2 weeks under validated storage conditions.
  • c.
    Resuspend cells at ∼2 × 10⁶ viable cells/mL.
  • d.
    Aliquot 1 mL of the cell suspension into each cryovial.
  • e.
    Place the cryovials in an isopropanol-based freezing container and freeze at −80°C for 12–24 h at a controlled cooling rate of approximately −1°C/min.
  • f.
    Transfer the cryovials to the vapor phase of liquid nitrogen for long-term storage.

Figure 5.

Culture and functional validation of MACS-enriched APCs

(A) Representative phase-contrast image of passage-0 MACS-enriched APCs at approximately 80%–90% confluence, demonstrating characteristic fibroblast-like morphology. Scale bar, 50 μm.

(B) Representative flow cytometry histogram showing Sca-1 expression in cultured passage-0 APCs at approximately 80%–90% confluence.

(C) Representative Oil Red O staining following adipogenic differentiation of the MACS-negative (flow-through) and APC-enriched fractions. The APC-enriched fraction exhibited greater intracellular lipid accumulation than the negative fraction.

(D) Relative mRNA expression of the adipogenic markers Adipoq, Pparg, and Fabp4 following adipogenic differentiation of the MACS-negative and APC-enriched fractions. Data are presented as mean ± SEM. ∗p < 0.05.

Expected outcomes

Successful execution of this protocol yields a heterogeneous stromal vascular fraction (SVF) containing stromal, immune, endothelial, and adipose progenitor cells (APCs). Following enzymatic digestion and filtration, properly processed samples should produce a single-cell suspension with minimal debris, and after centrifugation the SVF is observed as a compact, brownish pellet beneath a floating adipocyte/lipid layer. Cell yield and viability vary according to the adipose depot, tissue quality, and processing conditions. Preparations should meet the viability benchmarks described above before proceeding to downstream applications.

Following MACS enrichment, the APC fraction should show increased frequencies of lineage-negative Sca-1⁺PDGFRα⁺ cells, as confirmed by representative flow cytometric analysis (Figures 4B and 4C), although the degree of enrichment may vary depending on the tissue source and enrichment strategy. Quality control analyses should demonstrate efficient depletion of hematopoietic and endothelial cell populations together with enrichment of the expected APC marker profile. Under the recommended culture conditions, isolated APCs should adhere to tissue culture plastic within several hours of plating and display a characteristic fibroblast-like morphology. At passage 0, cells typically reach approximately 80%–90% confluence within 24–36 h when seeded at the recommended density (Figure 5A). Flow cytometric analysis at this stage demonstrates retention of Sca-1 expression in the cultured population (Figure 5B). Non-adherent cells are typically removed during the initial medium change, and APCs should maintain their proliferative capacity and characteristic morphology during early passages, although phenotypic drift may occur with extended culture.

Under adipogenic differentiation conditions, the APC-enriched fraction should accumulate abundant intracellular lipid droplets, as demonstrated by Oil Red O staining (Figure 5C), and exhibit significantly higher expression of the adipogenic markers Pparg, Fabp4, and Adipoq than the negative (flow-through) fraction (Figure 5D). Together, these findings provide functional validation that the MACS-enriched population possesses enhanced adipogenic differentiation potential and confirm that this protocol enriches for adipose progenitor cells rather than solely a surface marker-defined population. Because cell yield, viability, APC purity, and differentiation capacity may vary among adipose depots and experimental conditions, users are encouraged to establish these performance metrics for their own workflow.

Quantification and statistical analysis

Identity and purity assessment by flow cytometry

APC identity should be confirmed by flow cytometry using sequential gating of cells, singlets, viable cells, and lineage-negative (CD45⁻CD31⁻Ter119⁻) cells (Figure 4A), followed by identification of Sca-1⁺PDGFRα⁺ double-positive APCs (Figure 4B).^4,5,6,7,8 At a minimum, murine APC immunophenotyping should include CD45, CD31, and Ter119 as lineage exclusion markers together with Sca-1 and PDGFRα as APC enrichment markers, which also verify the efficiency of the MACS enrichment procedure9 (Figure 4B).⁹

Flow cytometry markers used for APC identification

Reagent	Application
Anti-CD45	Hematopoietic-lineage exclusion
Anti-CD31	Endothelial-lineage exclusion
Anti-Ter119	Erythroid-lineage exclusion
Anti-Sca-1	APC characterization
Anti-PDGFRα	APC characterization

Expected yields and viability benchmarks

SVF yield and viability vary according to adipose depot, donor characteristics, tissue quality, and processing conditions. Therefore, cell yield should be reported relative to the starting tissue mass (e.g., cells/g tissue), and users are encouraged to establish expected yields under their own experimental conditions. In our hands, freshly isolated SVF consistently exhibited approximately 90% viability before magnetic enrichment, whereas viability after MACS enrichment was typically 70%–80%. Post-isolation viability should generally exceed 70%, with ≥80% recommended for downstream cell culture and functional assays. Variability in yield, viability, and APC recovery should be documented together with digestion conditions, enrichment procedures, and tissue source.

Limitations

Despite providing a standardized and reproducible framework for APC isolation and culture, this protocol has several inherent limitations that should be considered when interpreting results.

Process-dependent heterogeneity of SVF and APC populations

A fundamental limitation of this protocol is that SVF and APC populations are not fixed biological entities but are highly dependent on processing conditions.⁴ Variations in enzymatic digestion (enzyme type, activity, and duration), tissue handling, washing stringency, and centrifugation parameters can significantly alter the cellular composition of the SVF.¹⁰ As a result, APC populations obtained using this workflow may differ between laboratories or experimental batches, even when nominally following the same protocol.

Enzymatic digestion-associated variability and cell stress

Enzymatic digestion is a critical but variable step that can influence both yield and cell viability.³ Overdigestion may reduce viability and damage cell surface markers, whereas underdigestion can result in incomplete tissue dissociation and reduced recovery. Additionally, enzyme activity may vary between lots, even when normalized by units, introducing variability across experiments.

Incomplete removal of non-target cell populations

Although MACS-based lineage depletion improves enrichment, it does not achieve the same level of purity as fluorescence-activated cell sorting (FACS). Residual hematopoietic, endothelial, or other stromal populations may persist following depletion, particularly when marker expression is low or heterogeneous. Consequently, downstream analyses may still reflect mixed cell populations.

Marker-based definition limitations of APCs

APCs lack a universally accepted, single definitive marker.⁴ The use of multi-parameter marker panels (e.g., Sca-1 and PDGFRα) improves enrichment but remains operational and context-dependent. Marker expression may vary by depot, species, and physiological state, and may shift during in vitro culture, limiting direct comparison across studies.

Culture-induced phenotypic drift

In vitro expansion can alter APC phenotype, including changes in surface marker expression, proliferative capacity, and differentiation potential. Early passage cells may more closely reflect in vivo biology, whereas extended culture can introduce selection bias toward specific subpopulations.

Sensitivity to pre-analytical handling conditions

Pre-analytical variables, including time from tissue harvest to processing, temperature fluctuations, and mechanical handling, can significantly impact cell viability and composition. Even minor deviations (e.g., delayed processing or inadequate cooling) may introduce variability that is difficult to control retrospectively.

Limitations in scalability and yield consistency

Although the protocol can be adapted for different input sizes, yield does not scale linearly with tissue volume. Larger samples may require modified digestion conditions or more complex workflows to maintain efficiency and viability. Additionally, depot-specific differences further limit reproducibility of absolute yields.

Potential impact of RBC removal strategies

Red blood cell (RBC) removal methods (e.g., washing versus lysis) can influence downstream cell composition and viability. While lysis improves purity, it may also introduce cellular stress if not tightly controlled, whereas insufficient removal may interfere with downstream applications such as flow cytometry or culture.

Technical limitations of MACS

MACS provides a rapid and scalable enrichment method but is inherently limited by antibody specificity, bead-binding efficiency, and column capacity. Non-specific binding, particularly in samples with low viability, may reduce enrichment specificity. Additionally, magnetic beads may remain associated with cells, which can interfere with certain downstream applications if not accounted for.

Batch-to-batch variability in reagents and materials

Variability in reagents (e.g., FBS, enzymes, antibodies) and consumables can introduce experimental variability. Even when standardized, differences between batches may affect cell behavior, growth kinetics, and differentiation outcomes.

Depot-specific variability

We performed representative validation using iWAT-derived SVF. Although the workflow is applicable to vWAT and BAT with optimization of digestion time, quantitative performance metrics, including SVF yield, viability, and post-MACS purity, may differ between depots because of depot-specific biological characteristics. Users are therefore encouraged to establish these parameters for their own experimental conditions.

Troubleshooting

Problem 1

Low SVF yield following enzymatic digestion (related to Steps 6–10).

Insufficient cell recovery may result from incomplete tissue digestion or loss during processing.

Potential solution

• •Ensure tissue is finely minced to increase enzyme accessibility.
• •Confirm enzyme activity (units/mL) and avoid relying solely on mg/mL.
• •Verify digestion temperature (37°C) and mixing conditions.
• •Avoid underdigestion. If intact tissue remains, continue digestion while monitoring tissue dissociation and remaining within the depot-specific digestion range specified in the protocol. Avoid extending digestion beyond the recommended depot-specific duration.
• •Minimize sample loss during transfers and aspiration steps.

Problem 2

Reduced cell viability after isolation (related to Steps 8–13).

Low viability may arise from overdigestion, prolonged RBC lysis, or excessive mechanical stress.

Potential solution

• •Avoid digestion beyond the recommended depot-specific duration, as prolonged exposure may reduce cell viability.
• •Strictly control RBC lysis duration (∼4 min on ice).
• •Use gentle pipetting to avoid shear stress.
• •Keep samples cold during all steps except digestion.
• •Process tissue immediately after collection to minimize degradation.

Problem 3

High red blood cell (RBC) contamination (related to Steps 2, 12–13).

Residual RBCs can interfere with downstream applications such as flow cytometry and culture.

Potential solution

• •Perform thorough tissue washing prior to digestion to reduce RBC load.
• •Optimize RBC lysis conditions (time and buffer volume).
• •Avoid under-lysis but also prevent prolonged exposure that reduces viability.
• •Consider repeating a short RBC lysis step if contamination persists.

Problem 4

Column clogging or slow flow during MACS (related to Steps 23–26).

Column blockage may occur due to debris, aggregates, or air bubbles.

Potential solution

• •Filter cell suspension through a 40 μm strainer before loading.
• •Degas MACS rinsing buffer to prevent bubble formation.
• •Avoid overloading columns and adhere to the recommended cell numbers.
• •Maintain continuous flow and do not allow columns to run dry.
• •If clogging occurs, collect cell suspension and reload onto a fresh column.

Problem 5

Low purity after MACS enrichment (related to Steps 21–27, 28–34).

Incomplete depletion or enrichment may result from suboptimal labeling or low marker expression.

Potential solution

• •Confirm cell viability (>70%) prior to labeling to reduce non-specific binding.
• •Ensure correct antibody/cocktail volume relative to cell number.
• •Maintain incubation at 2°C–8°C to improve specificity.
• •Perform a second column pass to increase purity (with potential yield trade-off).
• •Validate marker expression levels for the target population.

Problem 6

Poor cell attachment after seeding (related to Steps 35–37).

Cells may fail to adhere efficiently under suboptimal coating or seeding conditions.

Potential solution

• •Confirm proper coating of culture vessels (e.g., gelatin incubation and washing).
• •Ensure cells are seeded at an appropriate density.
• •Avoid excessive washing during the early attachment phase.
• •Use serum-containing medium to support adhesion.
• •Verify cell viability prior to plating.

Problem 7

Slow proliferation or inconsistent growth in culture (related to Steps 35–37).

Variability in growth may result from cell stress, suboptimal culture conditions, or donor variability.

Potential solution

• •Ensure consistent incubation conditions (37°C, 5% CO₂, humidified).
• •Use high-quality, consistent batches of FBS.
• •Avoid over-confluence or delayed passaging.
• •Standardize seeding density across experiments.
• •Minimize passage number to reduce phenotypic drift.

Problem 8

Unexpected variability in yield or marker expression (related to all steps).

Biological and technical variability may affect reproducibility.

Potential solution

• •Normalize yield to input tissue mass (cells per gram).
• •Record and report all critical parameters (time, temperature, enzyme activity).
• •Standardize tissue source, depot, and processing timeline.
• •Use consistent gating strategies in flow cytometry analysis.
• •Include biological replicates to account for variability.

Resource availability

Lead contact

Further information and requests for resources, reagents, and detailed protocol specifications should be directed to the lead contact, Dr. Marc G. Jeschke (jeschke@hhsc.ca), who will fulfill requests subject to reasonable availability.

Technical contact

Questions regarding experimental procedures, protocol implementation, troubleshooting, and technical aspects of the methodology should be directed to the technical contact, Ghazaleh Dadashizadeh (dadashig@mcmaster.ca).

Materials availability

No new unique reagents were generated in this study. All materials used are commercially available and are detailed in the key resources table. Additional information regarding reagent preparation and experimental conditions is available from the lead contact upon request.

Data and code availability

This study did not generate new datasets or custom code. All methodological details necessary to reproduce the protocol are provided within the manuscript.

Acknowledgments

This work was supported by the Juravinski Research Institute and grants from the National Institutes of Health (R01AG080040-01A1). F.K. is a recipient of the Canada Graduate Research Scholarship – Doctoral (Canadian Institutes of Health Research; # 210194) and receives support from McMaster’s Center for Metabolism, Obesity, and Diabetes Research graduate student award.

Author contributions

G.D. designed and developed the protocol, performed the experiments, analyzed the data, and wrote the manuscript. F.K. and S.W.-P. contributed to protocol optimization, experimental design, and manuscript editing. M.G.J. conceived the study, supervised the project, and provided critical revisions to the manuscript. All authors reviewed and approved the final version of the manuscript.

Declaration of interests

The authors declare no competing interests.