Isolation and immunofluorescence staining of Aspergillus fumigatus conidia-containing phagolysosomes.

The analysis of phagolysosomes within professional phagocytic cells is facilitated by their isolation. Here, we optimized a protocol for the isolation of intact phagolysosomes from macrophages infected with the spores of Aspergillus fumigatus. Purified phagolysosomes allow improved immunostaining, e.g., of phagolysosomal membrane proteins, or proteome analysis. For complete details on the use and execution of this protocol, please refer to Schmidt et al. (2020).

Before you begin

The protocol is optimized from the published work of Kyrmizi et al. (2018).

Phagocytic cells should be maintained according to the accepted protocols. Their viability should be higher than 90%, and confluency in flasks or wells should be around 80%.

For efficient uptake by phagocytic cells, fungal spores should be freshly harvested from mycelia.

Key resources table

Materials and equipment

For cultivation of RAW264.7 macrophages, the listed DMEM medium is recommended.

Step-by-step method details

Preparation of A. fumigatus conidia and macrophage cell line (RAW264.7)

For the isolation of phagolysosomes from macrophages seeding of a defined amount of macrophages in well plates and cultivation of A. fumigatus conidia until sporulation is necessary and described below.

Timing: 10 min

Revival of A. fumigatus conidia from cryopreservation

Use an inoculation loop to spread conidia on a petri dish with Aspergillus Minimal Medium agar (Pontecorvo et al., 1953)

Incubate conidia-plated agar plate for 5 days at 37°C

Timing: 30 min plus 3 days

Seed macrophage cell line

Preheat cell culture medium (see “Materials and equipment”), which should be stored at 4°C, to 37°C in a water bath

Prepare 2 × 25 cm2 cell culture flasks with 3 × 105 macrophages (revived from cryopreserved cell line) and 7 mL culture medium each

Incubate flasks for 3 days at 37°C and 5% (v/v) CO2

Timing: 1 h plus 1 day

Harvest conidia 5 days after mycelial growth and sporulation on agar plates

Slowly add 10 mL 0.9% (w/v) NaCl, 0.01% (v/v) Tween 20 solution to the petri dish

Use a T-formed spatula to scrap the conidia

Measure concentration of conidia by using a 1:100 dilution and counting with a Thoma chamber or another counting method

Harvest adherent macrophages with a confluency of 80%–90%

Pre-warm culture medium to 37°C in water bath

Remove the old medium from macrophages’ culture flask by aspiration

Add 3 mL of fresh pre-warmed medium

Use a cell scraper to scrape the macrophages from the base of the culture flask

Collect cell suspension in a 15 mL Falcon tube

Measure cell concentration by Thoma chamber or another counting system

Seed cells for infection with A. fumigatus conidia

Add 5 mL medium + 4 × 106 macrophages to each well of a 4-well plate (21,8 cm2/well)

Incubate cells 24 h at 37°C and 5% (v/v) CO2

Infection of macrophages with A. fumigatus conidia

Macrophages are incubated with a defined amount of conidia to allow phagocytosis and formation of a conidia-containing phagolysosome. The implementation of infection is explained in the following.

Timing: 3 h

Replace culture medium of the 4-well plate containing macrophages

Remove the old medium

Add 3 mL pre-warmed fresh medium to the cells

Phagocytosis of A. fumigatus conidia by macrophages

Infect macrophages using an MOI of 5; incubate for 30 min at 4°C to synchronize phagocytosis

Allow phagocytosis for 2 h at 37°C and 5% (v/v) CO2

Isolation of conidia-containing phagolysosomes

Formed phagolysosomes are isolated by the described cell lysis and Biocoll-separating process.

Timing: 3 h

Isolation of infected cells from plate

Pre-cool a tabletop centrifuge to 4°C

Remove old medium of the 4 wells of the infected macrophages by aspiration

Wash cells by adding 3 mL PBS per well and remove PBS

Harvest cells with cell scraper in 1.5 mL PBS per well

From each well, transfer cell suspension to a separate 1.5 mL Eppendorf tube

Centrifuge at 200 × g for 5 min at 4°C

Homogenization of cells

Discard supernatant

Resuspend cell pellet in 1 mL homogenization buffer (250 mM sucrose, 3 mM imidazole, pH 7.4) containing protease inhibitors

Lyse cells on ice by passing the cell suspensions 20 times per pellet through a 26G needle on a 1 mL syringe

CRITICAL: Slowly pass the cell suspension through a needle to avoid air bubbles in the tube. Passing times should not fall below 20 times to improve cell lysis.

Check cell breakage

Add 5 μL trypan blue to 5 μL cell suspension

Microscopic observation shows blue staining, which indicates disrupted cell membranes

Release of actin/myosin interaction

Incubate each cell homogenate with 300 μL of 10 mM ATP for 15 min with rotation at 4°C

Separation of phagolysosomes from cell debris

Split the 1 mL homogenate into portions of 500 μL; carefully overlay each 500 μL on top of 500 μL Biocoll (1.077 g/mL) in a reaction tube by using a 200 μL tip

CRITICAL: A small pipette tip avoids too fast layering of the homogenate onto Biocoll and improves separating process.

Centrifuge at 600 × g for 20 min at 4°C

Discard supernatant

Wash pelleted conidia-containing phagolysosomes twice with ice-cold 500 μL PBS with centrifugation in-between for 5 min, 600 × g, at 4°C

Resuspend pellet in 150 μL ice-cold PBS and collect all samples in one tube

Seed 20 μL suspension + 1 mL PBS or count phagolysosomes and place a certain amount on a coverslip in a 24-well plate and store at 4°C (possible for max. 1 day) to allow the phagolysosomes to attach onto the coverslip. Alternatively, isolated phagolysosomes can be lysed for Western Blot analysis.

Immunofluorescence staining of conidia-containing phagolysosomes

Timing: 6 h

Membrane-associated targets of isolated phagolysosomes can be stained with specific antibodies using immunofluorescence technology as described below.

Fixation of phagolysosomes

Slowly remove PBS from each well

Add 350 μL methanol/acetone (80%/20% (v/v)) or 3.5% (v/v) formaldehyde/PBS or 4% PFA for 15 min RT (depending on the target)

Wash three times with 350 μL PBS

Blocking and staining

Remove PBS

Add 350 μL 2% (w/v) BSA/PBS for 1 h

Remove blocking solution

Add 300 μL primary antibody diluted in blocking solutions as recommended for 1.5 h

Wash three times with PBS for 30 s

Remove PBS

Add 300 μL secondary antibody diluted in blocking solutions as recommended for 1 h

Wash three times with PBS for 30 s

Mount coverslips on a glass slide with mounting medium

Expected outcomes

For each condition, you should prepare 2 wells of a 4-well plate to isolate a sufficient number of phagolysosomes for immunofluorescence. If ∼20 μL of phagolysosome suspension are applied to a glass slide of a 24-well plate, it is possible to analyze 7–8 wells per condition in total. An example of an immunofluorescence staining of the vATPase V1 subunit on isolated phagolysosomes of RAW264.7 macrophages infected with A. fumigatus pksP conidia is shown in Figure 1.

Figure 1

Immunofluorescence staining of vATPase V1 subunit on isolated phagolysosomes of RAW264.7 macrophages infected with A. fumigatus pksP conidia

Anti-vATPase V1 antibody (Abcam, #ab73404) was used as primary antibody in a 1:500 dilution. Goat-anti-rabbit DyLight633 (Invitrogen, #35562) was used as secondary antibody in a 1:200 dilution. Arrows indicate isolated phagolysosomes. ∗ indicates intact macrophage.

The protocol can also be used for isolation of phagolysosomes from other cell lines and primary cells. Its suitability was confirmed for J774A.1 macrophage cell line and primary bone marrow-derived monocytes/macrophages from C57BL/6 mice.

Quantification and statistical analysis

Samples were visualized using a Zeiss LSM 780 confocal microscope and images were processed with the Zeiss ZEN software. Phagolysosomes with a positive signal for a specific target were counted and related to non-stained phagolysosomes. One hundred conidia-containing phagolysosomes per biological replicate were evaluated. The values represent the mean ± SD of three biological replicates. Additionally, immunofluorescence intensities could also be evaluated bioinformatically (Schmidt et al., 2020).

Limitations

The protocol leads to highly purified phagolysosomes; however, some cell debris or intact macrophages may remain in the suspension. A distinction of phagolysosomes from cell debris or intact cells is feasible by microscopy. A quantification of exclusively phagolysosomal proteins by Western blot proved to be more difficult.

Troubleshooting

Problem 1

The phagolysosomal suspension remains intact cells.

Potential solution

If effective isolation is uncertain, verification of damaged cells via trypan blue staining is necessary. Additional passing steps through a needle are recommended to further reduce the number of intact cells in the sample. Additionally, a careful overlaying improves phase separation between Biocoll and lysate. The use of a yellow tip for a pipetman facilitates the overlaying and improves the separation of phagolysosomes in the pellet and intact cells in the Biocoll-supernatant.

Problem 2

Phagolysosomes are not efficiently stained.

Successful staining of phagolysosomes with antibodies often depends on the fixation reagent, e.g., formaldehyde or methanol/acetone. If the immunofluorescence signal is weak or non-specific, other fixation methods or antibody concentrations can be assessed. For formaldehyde fixation, an addition of 0.3 M glycine to the blocking solution blocks free aldehyde groups and prevents non-specific binding.

Problem 3

Isolation of phagolysosomes only allows immunofluorescence of target structures on the outer membrane of phagolysosomes.

If your target of interest is expected on the inner phagolysosomal membrane, a permeabilization step with 1% (v/v) Triton X-100/PBS is recommended.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Axel A. Brakhage (axel.brakhage@leibniz-hki.de).

Materials availability

Any material generated in this study is available from the Lead Contact with a completed Materials Transfer Agreement.

Data and code availability

This study did not generate any unique datasets or code.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-vATPase V1 antibody	Abcam	#ab73404
DyLight633	Invitrogen	#35562
Chemicals
ATP	Sigma-Aldrich	A7699-1G
Biocoll	Merck	L6113
Bovine serum albumin	Sigma-Aldrich	A3294-50G
Mounting medium	Vector Laboratories	H-1000-10
Protease inhibitor	cOmplete, Roche	5892791001
Experimental models: cell lines
RAW264.7	ATCC	Cat#TIB-71
Experimental models: organisms/strains
Aspergillus fumigatus strain ATCC 46645 pksP	Jena Microbial Resource Collection (JMRC), HKI Jena	(Langfelder et al., 1998)
Software
BioRender	BioRender 2021	https://biorender.com/
Zeiss ZEN software 2.3	Zeiss	https://www.zeiss.de/

Reagent	Vendor/ manufacturer	Final concentration	Amount
DMEM medium (#11960044, high glucose)	Gibco	89%	178 mL
Fetal calf serum	GE Healthcare	10%	20 mL
Ultraglutamine	Gibco	1%	2 mL
Gentamicin sulfate 50 mg/mL	Gibco	27.5 μg/mL	110 μL
Total			200 mL