Cell culture material and conditions

The human microglia C20 cell line was donated by Dr. David Alvarez-Carbonell, Case Western Reserve University Cleveland, Ohio. Cells were cultured at 37 °C and 5% CO2 in complete medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, D6429), supplemented with 1 µM dexamethasone (PromoKine, PK-CA577-1042–1), 1% penicillin–streptomycin (Sigma-Aldrich, P4333), and 10% fetal bovine serum (FBS, Biowest, S181B). Hela cells were cultured at 37 °C, and 5% CO2 in Minimum Essential Medium (ThermoFisher Scientific, 31095-029) complemented with 1% penicillin–streptomycin (ThermoFisher Scientific, 15140-122), 1% L-glutamate 200 mM (ThermoFisher Scientific, 25030-024), and 10% fetal bovine serum (FBS, Biowest, S181B). Upon EV stimulation, the complete cell culture media supplemented with 10% FBS (Biowest, S181B) was replaced by the EV-depleted media containing 10% EV-depleted serum (System Bioscience, EXO-FBS-250A-1) to avoid bovine derived EV contamination.

Generation of HSPB8 CRISPR/Cas9 knock-out cell line

HSPB8 knock-out in HeLa cells was performed by CRISPR/Cas9 technology following the protocol described by Ran et al. [28]. In brief, at least two sgRNAs were designed using the online tool from the Broad Institute (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design), which cuts at least 30 amino acids after the start codon and which had the lowest number of off-target in the coding regions. The sgRNAs were ordered as phosphorylated primers from IDT (Integrated DNA Technologies) and cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 (plasmid #62988, Addgene). Constructs were transformed into DG-1 chemo competent bacteria, plated on agar plates, and sequence validated with Sanger sequencing. The plasmid with both the sgRNA of interest and the Cas9 was further used to transiently transfect naïve HeLa cells with Lipofectamine LTX with Plus Reagent (15338500 ThermoFisher Scientific). 24 h after transfection, the medium was refreshed with a medium containing puromycin (1 µg/mL) for 72 h to select the plasmid-containing cells. Surviving cells were serially diluted in a 96-well plate to obtain single colonies. Single clones were expanded, and the complete HSPB8 knock-out was verified for the presence of a premature stop codon by Sanger sequencing. The absence of the HSPB8 protein was verified by western blotting.

Generation of human oligodendroglia cell lines stably expressing HSPB8

The human oligodendroglia cell line stably expressing HSPB8 was generated as previously described by Irobi et al. [18]. In brief, constructs encoding the HSPB8 wild-type gene were created using the Gateway recombination technology, sequence validated, and PCR amplified (Invitrogen). The PCR products containing the HSPB8 ORF flanked by the attB recombination sites were then recombined into a pDONR221 vector and validated for the sequence. Next, pDONRs-HSPB8 constructs were transferred by recombination to the pLENTI6/V5-Dest (Invitrogen), generating constructs with the ORFs C-terminally fused to a V5 tag. According to a previously described method, the oligodendroglia cell line was produced by lentiviral transduction [29]. After infection, oligodendroglia was selected in DMEM supplemented with 10% FCS, 1% non-essential amino acids, 2 mm glutamine, and 100 µg/mL penicillin/streptomycin and blasticidin (3 µg/mL) for 3–4 weeks. The whole population of selected cells was pooled for further experiments. After selection, cells were cultured at 37 °C and 5% CO2 in complete medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, D6429), supplemented with 1% penicillin–streptomycin (Sigma-Aldrich, P4333), and 10% fetal bovine serum (FBS, Biowest, S181B).

Hollow fiber 3D EV production

Wild-type human oligodendroglia and stable HSPB8-overexpressing human oligodendroglia (OL-HSPB8) were used to produce native OL-EVs and modified OL-HSPB8-EVs. According to the manufacturer’s instructions, a medium-sized, hollow fiber culture cartridge with a 20 kDa molecular weight cut-off (Fibercell Systems, C2011) was pre-cultured. Cell lines were seeded and expanded in conventional culture flasks, harvested, and 2 × 108 cells were inoculated into the extra-capillary space of the hollow fiber cartridge and allowed to adapt to the bioreactor culture conditions for 11 days. Cells were cultured at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, D6429), supplemented with 1 × penicillin–streptomycin (Sigma-Aldrich, P4333), and 10% fetal bovine serum (FBS, Biowest, S181B). The pump flow rate was set following the manufacturer’s recommendations and media was refreshed after reaching 30% of the initial glucose concentration. Serum-free medium was used in the extra-capillary space, and EV-conditioned medium was collected 3 times a week.

Animals

C57BL/6 J mice were maintained according to the guidelines of the Belgian Law and the European Council Directive in the animal facility of the Biomedical Research Institute (BIOMED) at Hasselt University. The animals were housed on a 12 h light/dark cycle in standard cages with a controlled room temperature of 22 °C. Food and water were provided ad libitum. Experiments were conducted according to the European Community guiding principles on the care and use of animals and the approval of the Ethical Committee on Animal Research of Hasselt University.

Isolation and culturing of primary mixed neural culture cells

Primary mixed neural cultures were isolated from wild-type P3 mouse pups. Briefly, the cerebra were acutely isolated from decapitated pups and immediately transferred to ice-cold Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, D6429), supplemented with 1% penicillin–streptomycin (Sigma-Aldrich, P4333). Next, meninges were removed, and the hemispheres were transferred to fresh ice-cold Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich, D6429) containing 1% penicillin–streptomycin. The brains were mechanically homogenized, sieved through a 70 μm cell strainer, and centrifuged (300 × g, 10 min, 4 °C). The supernatant was discarded, and the pellet was resuspended in DMEM D6429 supplemented with 10% fetal bovine serum (FBS, Gibco, 26140-079), 10% horse serum (Gibco, 26050-088), and 1% penicillin–streptomycin (Sigma-Aldrich, P4333). Primary mixed neural cultures were obtained by seeding the cell suspension in poly-D-lysine pre-coated (PDL; 20 μg/mL, 1 h coating at 37 °C, Sigma-Aldrich, P6407-5MG) T75 culture flasks for 7 days before changing the medium.

EV isolation

Isolation and characterization of EVs were performed according to the MISEV 2018 guidelines [30]. In brief, the culture medium was collected and immediately pre-cleared for cells and debris by sequential centrifugation at 300 × g for 10 min, 2000 × g for 10 min, and 10,000 × g for 30 min. To pellet EVs, supernatants were ultra-centrifuged for 3 h at 115,000 × g. All centrifugation steps were performed at 4 °C using an Eppendorf 5804 R centrifuge or a Beckman Optima XPN-80 ultracentrifuge equipped with a Ti70 rotor. EV pellets were washed and collected in phosphate-buffered saline (PBS) and further purified by size-exclusion chromatography (SEC) using chromatography columns (Bio-Rad Laboratories, 732-1010) containing 10 mL Sepharose CL-2B beads (60–200 µm) (GE Healthcare,17-0140-01). The beads were sterilized using 70% ethanol (VWR chemicals, 84857.360) and rinsed with PBS. One mL volume of differential ultra-centrifuged EVs concentrate was loaded on the SEC column, followed by elution with PBS buffer to further purify the EVs. The eluate was collected immediately after EVs loading in 10 sequential fractions of 1 mL flow-through. Based on nanoparticle tracking analysis, collection volumes 4, 5 and 6 contained the highest EV concentration (data not shown). However, Böing et al. reported the presence of protein contaminants co-eluting from collection volume fraction 6 [31]. Therefore, fraction 6 was discarded, and EV-containing fraction 4 and 5 were pooled and up-concentrated using Amicon 10 k filters (Merck Millipore, UFC201024).

Quantification of EVs

Nanoparticle tracking analysis (NTA) was performed using the NanoSight NS300 system (Malvern Panalytical) equipped with a 532 nm laser to analyze EV concentration and size distribution. EV samples were diluted in PBS (Lonza, 17-516F) over a concentration range between 10 and 100 particles per frame. All settings were manually set at the beginning of the measurements and kept constant during all acquisitions: camera level, 14; camera gain, 1; pump rate, 80; viscosity, 1. A minimum of 3 recordings of 1 min per sample was analyzed by the NTA software 3.0 with default settings.

Transmission electron microscopy of purified EVs

OL-EV and OL-HSPB8-EV morphology was determined using transmission electron microscopy. Twenty microliter droplets of EVs suspended in PBS were placed on a Parafilm. Next, formvar-nickel TEM slots were placed on top of the EV droplets and allowed to adsorb for 60 min. Slots with adherent EVs were washed and fixed for 10 min with 2% glutaraldehyde. Subsequently, the slots were washed and transferred to droplets of 2% uranyl acetate for 15 min. Finally, the slots were embedded in 0.13% methylcellulose and 0.4% uranyl acetate. After drying, slots were examined with a Tecnai G2 Spirit Bio Twin transmission electron microscope (TEM, FEI/ThermoFisher, Eindhoven, The Netherlands)) at 120 kV.

Western blotting

EVs and cells were lysed for 30 min in ice-cold RIPA buffer (ThermoFisher, 89900), supplemented with protease (Roche, 05892970001) and phosphatase inhibitor (Roche, 04906845001). The lysate was cleared at 17,000 × g for 15 min. Next, protein concentrations were determined using Pierce BCA protein assay (ThermoFisher Scientific, 23227) according to the manufacturer’s protocol. Proteins were denatured at 95 °C for 5 min with 1 × sample buffer, loaded, and separated on a 12 or 15% SDS-PAGE gel. Subsequently, proteins were transferred for 1 h at 0.4 A on a polyvinylidene difluoride membrane (Immobilon®, IPVH00010) and blocked for 1 h with 5% milk powder in PBS (Lonza, 17-516F) containing 0.1% Tween 20 (VWR, MERC8.22185.0500). Finally, the membranes were incubated with a primary antibody overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies (Dako, Denmark) were used for 1 h at room temperature. Proteins were visualized using Pierce ECL Plus Western Blotting Substrate (ThermoFisher Scientific, 32132) for cell lysate or the WesternBright™ Sirius detection kit (Advansta, K-12043-D10) for EV lysate. Band intensities were determined by quantifying the mean pixel gray values using ImageJ software. The following primary antibodies were used: Exosome-anti-CD63 (Invitrogen, 10628D), Exosome-anti-CD9 (Invitrogen, 10626D), anti-Annexin A2 (Cell Signaling Technology, D1162), anti-Flotillin-1 (Cell Signaling Technology, D2V7J), anti-HSP70 (Santa Cruz Biotechnology, sc-32239), anti-Grp94 (Cell Signaling Technology, D6X2Q), anti-HSPB8/HSP22 (Cell Signaling Technology, 3059), anti-LC3B (Sigma-Aldrich, L7543), anti-BAG3 (Protein Tech, 10599-1-AP), anti-SQSTM1/P62 (Cell Signaling Technology, 5114), anti-β-actin (Santa Cruz Biotechnology, sc-47778), anti-ubiquitin (Cell Signaling Technology, 3936). The following secondary antibodies were used: polyclonal Rabbit Anti-Mouse Immunoglobulin/HRP (Dako, P0260) and polyclonal Goat Anti-Rabbit Immunoglobulin/HRP (Dako, P0448).

Real-time quantitative PCR

2 × 105 C20 cells were seeded in 6-well plates containing complete medium. After 24 h, the complete medium was replaced with EV-depleted medium, and cells were stimulated with 5 × 109 OL-EVs or OL-HSPB8-EVs for 24 h, followed by 100 ng/mL TNFα (Immunotools, 11343015) and 100 ng/mL IL1β (Immunotools, 11340015) activation for 24 h. As positive controls, PMA (100 ng/mL, 15 min), rapamycin (10 µM, 24 h), and staurosporine (2 µM, 3 h) were used. Subsequently, cells were harvested, and total RNA was prepared using the RNeasy Plus mini kit (Qiagen, 74106) according to the manufacturer’s instructions. RNA quality and quantity were determined using a NanoDrop spectrophotometer (Isogen Life Science, IJsselstein, The Netherlands). Further, RNA was reverse transcribed using the qScript™ cDNA SuperMix (Quanta Biosciences, 95048), and quantitative PCR was performed on a StepOnePlus™ RT-PCR System (Applied Biosystems, Halle Belgium). Fast SYBR Green (Applied Biosystems, 4385612), 10 µM forward and reverse primer mixture (Integrated DNA Technologies, Leuven, Belgium), and 10 ng RNA were used per reaction. Relative quantification of gene expression was accomplished using the ΔΔCt method. Expression levels were normalized using the most stable housekeeping genes; GAPDH and RPL13a as determined with geNorm. Primer sequences can be found in Additional file 1.

EV uptake by flow cytometry

C20 cells were seeded in 24-well plates at a density of 1 × 105 cells per well. Primary mixed neural culture cells were seeded in a 96-well plate at a density of 1.5 × 105 cells per well. After 24 h, cells were washed with PBS (Lonza, 17-516F) and activated with TNFα (100 ng/mL, Immunotools, 11343015) and IL1β (100 ng/mL, Immunotools, 1134O015) for 24 h to mimic inflammation. Next, cells were stimulated with 5 × 109 DiI-labelled EVs/mL in EV-depleted medium for 3 h at 37 °C to allow uptake. Equal volumes of OL-EVs and OL-HSPB8-EVs were labeled with 555 nm Vybrant DiI Cell-Labeling Solution (Life Technologies, 15704352) for 30 min at 37 °C. Unincorporated dye was removed by SEC. NTA was performed to determine EV concentrations. After incubation, cells were harvested using trypsin EDTA (Sigma-Aldrich, T3924) and centrifuged for 10 min at 300 × g. The supernatant was discarded, and cell pellets were resuspended in 500 µL PBS containing 4% FBS (Biowest, S181B). Single-cell mean fluorescent intensities were quantified at a wavelength of 555 nm by flow cytometry on a BD-LSRFortessa flow cytometer (BD Biosciences) using FACSDive software (BD Biosciences).

Chaperone activity assay

Naïve and HSPB8 KO HeLa cells were seeded in 6-well plates containing a complete medium. At 70% confluence, the complete medium was replaced by an EV-depleted medium, and cells were stimulated for 48 h either with 2 × 109, 5 × 109, 10 × 109 OL-EVs/mL, or OL-HSPB8-EVs/mL. After stimulation, cells were heat-shocked for 90 min at 42 °C, collected in ice-cold PBS, and pelleted by centrifugation. Subsequently, the pellets were resuspended in NP-40 lysis buffer (150 mM NaCl, 20 mM TrisBase, NP-40 0.05%, 1.5 mM MgCl2, Glycerol 3%, pH 7.4) supplemented with 1 M DTT and 1% Halt™ protease inhibitor (Thermo scientific, 78429), passed through a 23G 0.6 mm syringe 10 × and immediately centrifuged (16,100 × g, 15 min, 4 °C). The supernatant (soluble fraction) was collected, the pellet (insoluble fraction) was washed twice in ice-cold PBS, centrifuged (16,100 × g, 4 min, 4 °C), resuspended in equal volume used to lyse the cells of NP-40 buffer without DTT, and sonicated 10x (50% amplitude, 0.5 cycles) using a UP50H Ultrasonic Processor (Hielscher ultrasonics GmbH, Teltow, Germany). Next, from the soluble fraction, protein concentrations were determined using Pierce BCA protein assay (ThermoFisher Scientific, 23227) according to the manufacturer’s protocol. Proteins were denatured at 95 °C for 5 min with 1 × sample buffer, loaded, and separated on a 4–12% SDS-PAGE gel (NP0322BOX, Life Technologies). Running, transferring, blocking, and antibody incubation were performed as described previously.

Immunocytochemistry and confocal microscopy

Immunostainings were performed according to standard protocols. 5 × 104 C20 cells were seeded on a glass cover slide placed in a 24-well plate and submerged in complete medium. After 24 h, the complete medium was changed to EV-depleted medium, and cells were stimulated with either 5 × 109 OL-EVs/mL or 5 × 109 OL-HSPB8-EVs/mL. After 24 h, cells were activated with TNFα (100 ng/mL, Immunotools, 11343015) and IL1β (100 ng/mL, Immunotools, 11340015) to mimic inflammation. Autophagy was induced using 10 µM rapamycin (Enzo, ENZ-51031) for 24 h, and lysosomal degradation was inhibited using 10 µM chloroquine (Enzo, 51005-CLQ) for 18 h. Cells were fixed in ice-cold 100% methanol (VWR, 30847) for 15 min and permeabilized using 0.1% Triton X-100 (VWR, 437002A) in PBS (Lonza, 17-516F) for 15 min at 4 °C. Blocking was performed with 5% BSA (Life Sciences, A1324) diluted in PBS for 1 h. The primary LC3B antibody was incubated overnight at 4 °C, and the secondary antibody was incubated for 1 h, diluted in 5% BSA in PBS supplemented with 0.1% Tween 20 (VWR, MERC8.22185.0500) at room temperature. Nuclear staining was performed using DAPI (Thermo Fisher Scientific, 62248). Next, cells were mounted with a fluorescent mounting medium (Thermo Scientific, 9990402) and images were taken on a Zeiss LSM880 Airyscan laser scanning confocal microscope. All images used for quantification were acquired using identical fluorescence excitation and detection settings that avoided channel crosstalk. Three replicate wells were used per condition. An image analysis pipeline was designed in CellProfiler to automatically quantify the number of punctate structures (LC3B, mean fluorescence intensity) per individual cell. In short, nuclei were segmented based on the DAPI channel (Adaptive thresholding Otsu method) and used as seeds to segment the cells (Propagation method with global Otsu thresholding). Punctate structures were segmented in the appropriate channel using the global MoG thresholding channel. Standard CellProfiler modules were used to relate the different masks, measure shape, and intensity parameters, and create output tables.

CYTO ID autophagy assay

Autophagy induction was determined by using the CYTO-ID Autophagy Detection Kit (Enzo, ENZ-51031) according to the manufacturer’s protocol. Briefly, 1 × 105 C20 cells or 2 × 105 primary mixed neural culture cells were seeded in 24-well or 96-well plates containing complete medium, respectively. The next day, the complete medium was changed to EV-depleted medium, and cells were stimulated for 48 h with either 5 × 109 OL-EVs/mL or 5 × 109 OL-HSPB8-EVs/mL. At 24 h after EV stimulation, cells were activated for 24 h by a mixture of 100 ng/mL TNFα (Immunotools, 11343015) and 100 ng/mL IL1β (Immunotools, 11340015) to mimic inflammation or cells were stimulated with 10 µM rapamycin (Enzo, ENZ-51031) for 24 h to induce autophagy. Lysosomal degradation was blocked using 10 µM chloroquine (Enzo, 51005-CLQ) for 18 h. After incubation, cells were trypsinized, centrifuged for 10 min at 300 × g, PBS-washed, again centrifuged, and resuspended in 0.5 mL assay buffer. Subsequently, cells were stained with the CYTO-ID green detection dye for 30 min at 37 °C in the dark. After staining, the cells were pelleted at 300 × g for 10 min and resuspended in 0.5 mL assay buffer. Single-cell mean fluorescent intensities were quantified at a wavelength of 488 nm by flow cytometry on a BD-LSRFortessa flow cytometer (BD Biosciences) using FACSDive software (BD Biosciences).

Transmission electron microscopy assessment of autophagy

Human microglia C20 cells (5 × 104) were grown in 8-well chambered Permanox slides (Nunc, Lab-Tek, C7182-1PAK) and stimulated with 5 × 109 OL-EVs/mL or OL-HSPB8-EVs/mL in 400 µL EV-depleted medium. As a control, cells were stimulated with 10 µM rapamycin (Enzo, ENZ-51031) for 24 h to induce autophagy. Lysosomal degradation was blocked using 10 µM chloroquine (Enzo, 51005-CLQ) for 18 h. After stimulations, C20 cells were fixed in 0.1 M sodium cacodylate-buffered, pH 7.4, 2.5% glutaraldehyde solution for 2 h at 4 °C and washed in 0.1 M sodium cacodylate, pH 7.4 (Sigma-Aldrich, 6131–99-3) containing 7.5% saccharose (Sigma-Aldrich, 57-50-1). Next, cells were incubated for 1 h in 1% OsO4 solution (Sigma-Aldrich, 20816-12-0). After dehydration in an ethanol gradient, cells were embedded in EM-bed812 (Electron Microscopy Sciences, EMS14120). Ultrathin sections were stained with lead citrate and samples were examined in a Tecnai G2 Spirit Bio Twin Microscope (FEI, Eindhoven, The Netherlands) at 120 kV. Quantification of autophagic vesicles was achieved by manually counting vesicles. Images were blinded and randomly taken at a magnification of 26,000 × in the cytoplasm of the cells. Autophagic vesicles were identified based on ultrastructural characteristics as described by Ylä-Anttila et al. [32].

ROS-DCFH assay

ROS activity was measured by seeding 1 × 105 C20 cells or 2 × 105 primary mixed neural culture cells in 24-well or 96-well plates containing complete medium, respectively. The next day, cells were stimulated with 5 × 109 OL-EVs/mL or 5 × 109 OL-HSPB8-EVs/mL in an EV-depleted medium. Subsequently, cells were left non-activated, activated with 100 ng/mL TNFα (Immunotools, 11343015) and 100 ng/mL IL1β (Immunotools, 11340015) for 24 h or 100 ng/mL PMA (Merck, P1585) for 15 min. After stimulations, cells were harvested using trypsin, centrifuged, and stained with 10 µM 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Enzo, ALX-610–022-M050) solution for 30 min at 37 °C in the dark. Subsequently, cells were pelleted, washed, and resuspended in PBS containing 4% FBS (Biowest, S181B). Single-cell mean fluorescent intensities were quantified at a wavelength of 488 nm by flow cytometry on a BD-LSRFortessa flow cytometer (BD Biosciences) using FACSDive software (BD Biosciences).

JC-1 mitochondrial membrane potential assay

The JC-1 Mitochondrial Membrane Potential Assay Kit (BD Biosciences, 551302) was used according to the manufacturer’s protocol. In brief, 1 × 105 C20 cells or 2 × 105 primary mixed neural culture cells were seeded in 24-well or 96-well plates containing complete medium, respectively. After 24 h, the complete medium was changed to EV-depleted medium, and cells were stimulated with 5 × 109 OL-EVs/mL or OL-HSPB8-EVs/mL, followed by TNFα (100 ng/mL, Immunotools, 11343015) and IL1β (100 ng/mL, Immunotools, 11340015) activation for 24 h or PMA (100 ng/mL, Merck, P1585) stimulation for 15 min. Subsequently, cells were harvested, centrifuged, PBS-washed, again centrifuged, and suspended in 0.5 mL assay buffer. Next, cells were stained with the JC-1 green detection dye for 30 min at 37 °C in the dark. After centrifugation, the supernatant was discarded, and the pellet was resuspended in 0.5 mL assay buffer. Single-cell mean fluorescent intensities were quantified at a wavelength of 488 nm and 594 nm by flow cytometry on a BD-LSRFortessa flow cytometer (BD Biosciences) using FACSDive software (BD Biosciences).

Annexin V phosphatidylserine (PS) apoptosis assay

Phosphatidylserine (PS) exposure was explored using the PS-binding protein Annexin V conjugated to a fluorescent FITC label to determine early apoptosis. 2 × 105 primary mixed neural culture cells or 1 × 105 C20 cells were seeded in 96-well or 24-well plates containing complete medium, respectively. The next day, the complete medium was switched to EV-depleted medium, and cells were stimulated with 5 × 109 OL-EVs/mL or OL-HSPB8-EVs/mL for 24 h, followed by TNFα/IL1β (100 ng/mL, Immunotools 11343015, 11340015) activation for 24 h. Apoptosis was induced using 2 µM staurosporine (Enzo, ALX-380-014-M001) for 3 h. Cells were collected, centrifuged, and resuspended in 100 µL of assay buffer (Invitrogen, PNN1001). Per reaction, 5 µL Annexin V-FITC (Invitrogen, 11-8005-74) and 2.5 µL 7AAD dye (BD Pharmingen, 51-68981E) were incubated for 15 min in the dark at room temperature. Next, cells were centrifuged, washed, and resuspended in 1 × assay buffer. Single-cell fluorescent intensities were quantified by flow cytometry on a BD-LSRFortessa flow cytometer (BD Biosciences) using FACSDive software (BD Biosciences).

Statistical analysis

Statistical analyses were performed using Student’s two-tailed t-test or one-way ANOVA unless noted otherwise. Data were analyzed using the GraphPad Prism software and presented as the mean ± SEM. Values of P < 0.05 were considered statistically significant.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Disclaimer:

This article is autogenerated using RSS feeds and has not been created or edited by OA JF.

Click here for Source link (https://www.biomedcentral.com/)