Detection of Autophagy in Human Peripheral Blood Mononuclear Cells Using Guava^® Autophagy and Flow Cytometry

Materials and reagents

Biological materials

1. Primary human peripheral blood mononuclear cells (PBMCs) from volunteers

Reagents

1. S-Monovette^® K3 EDTA 9 mL (SARSTEDT, catalog numbers: 02.1066.001)

2. Ficoll-Paque^TM PLUS density gradient media (Cytiva, catalog number: 17-1440-03)

3. RPMI medium 1640 (1×) without phenol red (Gibco^TM, catalog number: 11835063)

4. Chloroquine diphosphate (TOCRIS, catalog number: 4109)

5. Dimethyl sulphoxide (DMSO) ≥ 99.5%, BioScience grade (Carl Roth, catalog number: A994.2)

5. Guava^® Autophagy LC3 Antibody-Based Detection kit (Cytek^®, catalog number: FCCH100171)

6. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957)

7. Di-sodium hydrogen phosphate dodecahydrate (Na₂HPO₄·12H₂O) (Carl Roth, catalog number: N350)

8. Potassium dihydrogen phosphate (KH₂PO₄) (Carl Roth, catalog number: 3904)

9. Potassium chloride (KCl) (Carl Roth, catalog number: 6781)

10. Water, molecular biology grade (VWR, catalog number: VWRL0201-0500)

11. Acridine orange/propidium iodide stain (Logos, catalog number: F23001)

Solutions

1. Physiological saline solution (0.9% NaCl) (see Recipes)

2. 1× Phosphate buffered saline (PBS) (see Recipes)

3. 1× Assay buffer (see Recipes)

4. Autophagy reagent B (see Recipes)

5. LC3 staining solution (see Recipes)

Recipes

1. Physiological saline solution (0.9% NaCl)

Reagent	Final concentration	Quantity or volume
NaCl	0.9% (w/v)	9.0 g
Deionized water	n/a	to 1,000 mL
Total	n/a	1,000 mL

Autoclave and store at room temperature.

2. 1× PBS

Reagent	Final concentration	Quantity or volume
NaCl	8.0% (w/v)	80.0 g
KCl	0.2% (w/v)	2.0 g
Na2HPO4·12H2O	1.78% (w/v)	17.8 g
KH2PO4	0.24% (w/v)	2.4 g
Deionized water	n/a	to 1,000 mL
Total	n/a	1,000 mL

Adjust pH value to 7.4. Autoclave 10× PBS; for 1× PBS, dilute 10× stock 1:10 with deionized water (50 mL of 10× PBS + 450 mL of deionized water), autoclave, and store at room temperature.

3. 1× Assay buffer

Reagent	Final concentration	Quantity or volume
5× Assay buffer (from Guava®)	1×	55 mL
Deionized water	n/a	220 mL
Total	n/a	275 mL

Dilute and then sterilize by filtration and store at 4 °C for up to one year.

4. Autophagy reagent B

Reagent	Final concentration	Quantity or volume
Autophagy reagent B (10×)	1×	1 mL
Water, molecular biology grade	n/a	9 mL
Total	n/a	10 mL

Aliquot and store at 4 °C for up to one year.

5. LC3 staining solution

Reagent	Final concentration	Quantity or volume
20× anti-LC3/FITC antibody	1×	62.5 µL
1× assay buffer	n/a	1,187.5 µL
Total	n/a	1,250.0 µL

Prepare immediately before use; do not store. Protect from light. All listed ingredients are part of the Guava^® Autophagy LC3 Antibody-Based Detection kit.

Laboratory supplies

1. Leucosep^TM with porous barrier, 50 mL (Greiner Bio-One GmbH, catalog number: 227290)

2. Centrifuge tubes, 15 and 50 mL (SARSTEDT, catalog numbers: 62.554.502, 62.547.254)

3. Reaction tubes, 1.5 and 2 mL (SARSTEDT, catalog numbers: 72.706, 72.695.500)

4. LUNA 2-channel cell counting slides (Logos, catalog number: L12002-LG)

5. Pipette tips, 20, 200, and 1,000 μL (SARSTEDT, catalog numbers: 701.114.210, 70.3030, and 70.3050)

6. Serological pipettes, 5, 10, and 25 mL (SARSTEDT, catalog numbers: 86.1253.001, 86.1254.001, and 86.1685.001)

7. Syringe filter, Filtropur S, pore size: 0.2 μm (SARSTEDT, catalog number: 83.1826.001)

8. Corning^® 96-well clear V-bottom TC-treated microplates (Corning, catalog number: 3894)

Equipment

1. Pipettes: Eppendorf Research^® Plus 10 μL, 20 μL, 200 μL, 1,000 μL (Eppendorf, catalog numbers: 3123000020, 3123000039, 3123000055, 3123000063)

2. Multichannel microliter pipette Transferpette^®, 20–200 µL (BRAND GMBH + CO.KG, catalog number: 9280173)

3. Pipetting aid: PipetBoy acu 2 (Integra Biosciences, catalog number: 155 000)

4. Luna^TM fl Dual Fluorescence Cell Counter (Logos, model: L20001-LG)

5. Water bath (GFL, catalog number: 1003)

6. Microcentrifuge Eppendorf 5424 (Eppendorf, catalog number: 05-400-005)

7. Swing-out rotor centrifuge Eppendorf 5804 R (Eppendorf, catalog numbers: 5805000010, 5804709004)

8. Water purification system ELGA^® PURELAB flex 3 (Veolia, catalog number: PF3XXXXM1)

9. pH meter FE20 FiveEasyTM (Mettler Toledo, catalog number: 30266626)

10. Heat treatment IRAcubator (Von Ardenne Institut für Angewandte Medizinische Forschung GmbH)

11. Incubator at 37 °C with 5% CO₂, 90% humidity (HERA Cell 240, catalog number: 2510-413-01)

12. BD FACSVerse^TM with 488 nm laser (BD Biosciences, catalog number: 651155)

Software and datasets

1. BD FACSSuite (BD Biosciences, v1.0.6), license needed

2. GraphPad Prism (GraphPad Software, Inc., v. 10.4.2), license needed

Procedure

A. PBMC isolation

1. Warm the Ficoll-Paque^TM PLUS separation medium to room temperature (RT), protected from light, and fill the Leucosep^TM tube with 15 mL separation medium. Centrifuge at 1,000× g for 1 min at RT. The separation medium is now located below the porous barrier.

Note: All centrifugation steps are carried out at RT.

2. Dilute an appropriate volume of anticoagulated human blood (collected in 9 mL of K3 EDTA S-Monovette^®) at a ratio of 1:2 with 0.9% NaCl solution in a separate centrifuge tube. Transfer the diluted blood carefully into the prepared Leucosep^TM tube (at least 15 mL and at most 30 mL).

3. Centrifuge at 1,000× g for 10 min in a swing-out rotor with brake switched off.

Figure 2. Primary human peripheral blood mononuclear cell (PBMC) isolation using Leucosep^TM tubes. On the left side, the porous barrier separates the Ficoll-Paque^TM PLUS (bottom) and the diluted anticoagulated blood (top). On the right side, after centrifugation, the sequence of layers occurs as follows (from top to bottom): plasma > enriched cell fraction (PBMCs) > Ficoll-Paque^TM PLUS > porous barrier > Ficoll-Paque^TM PLUS > erythrocytes and granulocytes.

4. Collect and discard approximately 10 mL of plasma from the top (Figure 2), leaving a minimum remnant of 5–10 mm above the interphase, to prevent contamination of the enriched cells with platelets and facilitate harvesting of the enriched cell fraction.

5. Harvest the enriched cell fraction (lymphocytes/PBMCs) using a 1,000 μL Eppendorf pipette. The porous barrier effectively prevents recontamination with pelleted erythrocytes and granulocytes (for detailed information, see the manufacturer's instructions or refer to [8]).

6. Wash the cells with 15 mL of 1× PBS and subsequently centrifuge at 250× g for 10 min with the brake switched back on.

7. Repeat the washing step twice, resuspending the cell pellet in 10 mL of PBS each time.

8. Determine the cell count using acridine orange/propidium iodide stain and the Luna^TM fl Dual Fluorescence Cell Counter.

B. Cell seeding and treatment

1. Prepare six wells per plate (control plate, treatment plate) with 500,000 cells/well in a 96-well plate (V-bottom) in 200 μL of RPMI medium (Figure 3a.) or 200 μL of RPMI medium with 40 μM chloroquine (CQ; diluted with RPMI medium from 600 µM CQ in DMSO) (Figure 3b.) in triplicate (Figure 3).

Figure 3. Plate layouts. Both the control plate (left) and the treatment plate (right) contain three wells with RPMI medium (a.) for measurement and six wells with RPMI medium with 40 µM chloroquine (b.) as a positive control.

2. The IRAcubator should be preheated to 39 °C.

3. Place the control plate in a humidified incubator at 37 °C and 5% CO₂ for 1 h.

4. Place the treatment plate in the IRAcubator at 39 °C for 1 h (Figure 3) (for detailed information, refer to [7]).

C. Guava^® autophagy detection

1. After incubation with CQ and/or treatment, centrifuge the plates at 300× g for 5 min. Discard the supernatant by turning the plate(s) over onto laboratory tissue paper.

2. Resuspend the pellets in 100 μL of 1× assay buffer, then centrifuge the plates again at 300× g for 5 min.

Note: Allow all assay components to reach RT.

3. After centrifugation, add 100 μL of autophagy reagent B directly to each well, then immediately centrifuge again at 300× g for 5 min.

Caution: Do not discard the supernatant; otherwise, the concentration of autophagy reagent B will be incorrect.

4. Discard the supernatant by turning the plate(s) over onto laboratory tissue paper. Resuspend the pellets in 100 μL of LC3 staining solution and leave to incubate for 30 min at RT in the dark.

Note: All subsequent steps are carried out in the dark.

5. After incubation, centrifuge at 300× g for 5 min and discard the supernatant by turning the plate(s) over onto laboratory tissue paper.

6. Wash the samples once with 100 μL of 1× assay buffer to remove any residual or unbound antibody.

7. Centrifuge at 300× g for 5 min and discard the supernatant by turning the plate(s) over onto laboratory tissue paper.

8. Resuspend the pellets in 100 μL of 1× assay buffer and then leave the plates for 10 min at RT in the dark, directly before measurement.

9. Analyse samples via flow cytometry with BD FACSVerse^TM for data acquisition.

D. Data acquisition

1. The analysis of antibody-based LC3 detection is carried out via flow cytometry using a BD FACSVerse^TM instrument. Samples are measured directly in 96-well plates using a universal loader.

Caution: Make sure to use the right plate geometry (Corning^® standard conical bottom).

2. After manually gating the lymphocytes using forward scatter (FSC) and side scatter (SSC), the median fluorescence intensity (MFI) is measured (Figure 4).

3. A total of 10,000 events are gated, and the FITC-A median is defined as the MFI and used for subsequent analysis.

Figure 4. Forward scatter/side scatter (FSC/SSC) and histogram plots for the gating process (LC3 detection). As only a subpopulation (lymphocytes) of the total measured cells is relevant for LC3 detection, the corresponding population is manually gated in the FSC and SSC plots (left). Based on this population, the number of FITC-positive events is measured in the histogram (right).

Data analysis

As previously stated, three technical replicates should be carried out for each condition. Outliers can be excluded; we recommend defining them as the mean plus/minus three standard deviations.

Statistical analyses can be performed using any version of Prism (GraphPad Software, Inc.) or your preferred statistical software (e.g., R).

Validation of protocol

This protocol (or parts of it) has been used and validated in the following research articles:

1. Hochecker et al. [9] Heat treatment in health and disease: How water-filtered infrared-A (wIRA) irradiation affects key cellular mechanisms in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients compared to healthy donors. Journal of Thermal Biology (Figure 2).

2. Hochecker et al. [10] Heat vs. Fatigue: Hyperthermia as a Possible Treatment Option for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). International Journal of Molecular Sciences (Figure 1).

General notes and troubleshooting

General notes

1. When investigating the direct effect of an intervention on the organism (see [10]), the “before” and “after” measurements must be carried out directly, meaning there should be no delay between the two samples, and the samples must be measured independently, even if this means the measurements are taken on different days.

Troubleshooting

Problem 1: The 10× assay buffer shows signs of precipitation.

Possible cause: The 10× assay buffer was stored at too low a temperature.

Solution: Place the bottle in a 37 °C water bath, shaking or swirling occasionally. If the precipitate does not completely dissolve, leave the 10× assay buffer at RT overnight.

Problem 2: The lymphocyte population is outside of the predefined population gate.

Possible cause: Due to donor variability, the population has slightly shifted in the FSC/SSC plot.

Solution: Manually adjust the gate to encompass the entire lymphocyte population.

Problem 3: The CQ-treated cells show no offset in the histogram plot.

Possible cause: Either the concentration of CQ was not suitable for this cell type, or the incubation period was too short.

Solution: Determine the appropriate concentration of CQ via titration for the intended cell type or increase the incubation time with CQ prior to the assay.

Acknowledgments

Author contributions: Conceptualization, M.S., B.H., K.M., A.M., and J.B.; Investigation, M.S., B.H.; Writing—Original Draft, M.S.; Writing—Review & Editing, M.S., B.H., K.M., and J.B.; Funding acquisition, J.B.; Supervision, K.M., B.H., and J.B. The authors thank Dr Stefan Pieper and Dr Achim Schneider for providing the blood samples. The authors also thank Dr Alexander von Ardenne and Noah Molinski for the construction of the IRAcubator and good cooperation. This work was funded by the Professor Manfred von Ardenne Forschungsförderungsgesellschaft e.V. This work was also funded by the Baden-Württemberg Ministry for Science, Research and Art (MWK Baden-Württemberg) as part of the Kooperatives Promotionskolleg (KPK) InViTe2 Sigmaringen/Konstanz by financing Melanie Scherer as a doctoral student, and in which Barbara Hochecker is an associated PhD student. This protocol was used in Hochecker et al. [9] and Hochecker et al. [10].

Competing interests

The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests: Melanie Scherer reports that financial support was provided by the MWK Baden-Württemberg. Barbara Hochecker reports that financial support was provided by Professor Manfred von Ardenne Forschungsförderungsgesellschaft e.V.

Ethical considerations

This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Landesärztekammer Baden-Württemberg (F-2013-066#A2, 31 January 2023). Informed consent was obtained from all subjects.

References

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