Overview
This video demonstrates a small interfering RNA (siRNA)-based technique to interfere with autophagic flux. A siRNA targeting FIP200 mRNA is introduced into monocyte-derived immature dendritic cells (iDCs) using electroporation, causing the knockdown of FIP200 expression and leading to the inhibition of nuclear lamin disruption via autophagy. Upon adding herpes simplex virus (HSV), the nuclear egress of viral progeny is inhibited due to the absence of lamin disruption via autophagic flux.
Protocol
Monocyte-derived DCs were generated from leukapheresis products of healthy donors. For this, a positive vote from the local ethics committee has been obtained (reference number 4556). The experiments of the present study were performed in accordance with the recommendations of the ethics committee of the "Friedrich-Alexander-Universität Erlangen-Nürnberg" (reference number 4556). All donors approved a written informed consent, including the accordance with the Declaration of Helsinki.
1. Generation and handling of immature dendritic cells (iDCs) and mature dendritic cells (mDCs)
- Isolate human peripheral blood mononuclear cells (PBMCs) from leukoreduction system chambers (LRSCs) as previously described. Avoid cryopreservation of PBMCs and use them directly upon isolation to obtain higher DC yields.
- Generate human DCs from PBMCs of different healthy donors in T175 cell culture flasks as previously described. Briefly, use 350-400 millions of PBMCs in 30 mL of DC medium (RPMI 1640 without L-glutamine, 1% (v/v) Human Serum Type AB (AB-serum), 100 U/mL penicillin, 100 mg/mL streptomycin, 0.4 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)) per cell culture flask for isolation of monocytes by adherence. After 1 h, wash off nonadherent fraction using RPMI 1640. Add fresh DC medium supplemented with 800 U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 250 U/mL interleukin-4 (IL-4), and incubate for 3 days.
- On day 3 post adherence, add 5 mL of fresh DC medium containing GM-CSF and IL-4 with a final concentration of 400 U/mL and 250 U/mL per cell culture flask, respectively, for DC differentiation.
- To harvest iDCs, gently rinse loosely-adherent iDCs from the bottom of the cell culture flask, on day 4 post adherence. Repeat this step 2 times. For generation of mDCs, add maturation cocktail composed as follows: GM-CSF (final concentration: 40 U/mL), IL-4 (final concentration: 250 U/mL), interleukin-6 (IL-6, final concentration: 1000 U/mL), Interleukin-1 beta (IL-1β, final concentration: 200 U/mL), Tumor Necrosis Factor alpha (TNF-α, final concentration: 10 ng/mL), prostaglandin E2 (PGE2; final concentration: 1 μg/mL).
- Six days post adherence (two days post induction of maturation using a cytokine cocktail), rinse mDCs from the bottom of the cell culture flask. Repeat this step two times.
NOTE: Immature and mature DCs can be sequentially generated from identical donors in 1 cell culture flask. To do so, (i) separate the appropriate number of iDCs and (ii) induce maturation of the remaining cells in the flasks using the cytokine cocktail listed in step 1.2.2.
- Transfer iDCs or mDCs in the respective cell culture medium into 50 mL tubes. Harvest the cells via centrifugation at 300 x g for 5 min.
- Gently resuspend (=wash) DCs in 5-10 mL of RPMI 1640 per cell culture flask. Combine respective DC suspensions in one tube.
- Define the cell number using a counting chamber or an alternative method. Avoid temperature alterations when handling iDCs, to reduce the risk of phenotypic changes.
2. Flow cytometric analyses to monitor the phenotypic maturation status
- Transfer iDCs or mDCs (0.5 x 106) from step 1.3.1 into a 1.5 mL tube. Harvest the cells via centrifugation at 3390 x g for 1.5 min. Wash the cells once with FACS buffer (PBS supplemented with 2% fetal calf serum (FCS)).
- Resuspend the cells in 100 µL of antibody staining solution (FACS buffer) containing specific fluorochrome-labeled antibodies against defined surface molecules.
- Use the following antibodies to verify purity (CD3- fluorescein isothiocyanate [FITC], CD14- phycoerythrin [PE]) as well as maturation status of DCs (CD80-PacBlue [Pacific Blue]/-PE- cyanine5 [Cy5], CD11c-PE-Cy5, CCR7-PE-Cy7, CD83-APC, CD86-PE, MHCII-APC-Cy7).
- Prepare one unstained sample in 100 µL of FACS buffer as a control.
- Stain the cells on ice in the dark for 30 min.
- Subsequently wash the cells two times in 1 mL of FACS buffer and centrifuge at 3390 x g for 1.5 min.
- Finally, resuspend the cells in 200 µL of FACS buffer supplemented with 2% paraformaldehyde (PFA) and analyze the cells by flow cytometry. Fixed cells can be stored at 4° C in the dark up to 2 days.
3. Interference of HSV-1-induced autophagic flux via electroporation of iDCs using FIP200-siRNA
NOTE: The present protocol for siRNA electroporation was modified from Prechtel et al. (2007) and Gerer et al. (2017).
- Transfer iDCs (12 x 106) at day 3.5 post adherence into a 50 mL tube. Subsequently, centrifuge the cells at 300 x g for 5 min and discard the supernatant. In parallel, perform flow cytometric analysis to monitor the maturation status as described in step 2 (Use Life/Dead violet instead of CD80-PacBlue).
- Gently wash iDCs in 5 mL of OptiMEM without phenol red and centrifuge the cells at 300 x g for 5 min. Discard the supernatant and gently resuspend iDCs in 200 µL of OptiMEM without phenol red, adjusting a cell concentration of 6 x 106/100 µL. Do not place the cells on ice and avoid temperature alterations. Move on quickly and avoid long incubation periods of iDCs in OptiMEM without phenol red.
- Transfer either 75 pmol of FIP200-specific siRNA or 75 pmol of scrambled siRNA, as a control, into 4 mm electro cuvettes and add 100 µL (6x106 cells) of the cell suspension. Directly pulse iDCs using the electroporation apparatus I, applying the following settings: 500 V for 1 ms.
- Prior to the experimental procedure, prepare siRNA suspensions according to the manufacturer's instruction, aliquot and store them at -20 °C. Thaw and keep them on ice when using these siRNAs for electroporation. Before electroporating the samples, perform a test pulse.
- After electroporation, directly transfer iDC into 6-well plates with fresh pre-warmed DC medium (supplemented with 40 U/mL of GM-CSF and 250 U/mL of IL-4). Seed the cells at a final concentration of 1 x 106/mL and place them into an incubator. Do not rinse the cells out of the electro cuvette.
- After 48 h, first examine the morphology of electroporated iDCs microscopically. Then, harvest the cells using a cell scrapper and transfer them into 15 mL tubes. Rinse the wells with 1 mL of PBS supplemented with 0.01% ethylenediaminetetraacetic acid (EDTA) and transfer the solution in the respective tubes.
- Subsequently, split 6 x 106 iDCs per siRNA condition as described in the next steps.
- Use 0.5 x 106 cells to assess the maturation status and cell viability as described in step 2. Use the following antibodies: CD11c-PE-Cy5, CCR7-PE-Cy7, CD83-APC, MHCII-APC-Cy7 and Life/Dead violet.
- Use 1 x 106 cells for Western blot analyses to verify FIP200-specific knockdown efficiency. Transfer and harvest the cells into a 1.5 mL safelock tube by centrifugation at 3390 x g for 1.5 min. Prepare cell lysates and perform Western blot analyses.
- Use the remaining cells (4.5 x 106) for HSV-1 infection experiments. For each experimental condition, transfer 2.25 x 106 iDCs into 2 mL tubes and either infect them with HSV1 at an multiplicity of infection (MOI) of 2 or add MNT buffer as a mock control. Perform the infection as described.
- At 20 h post infection (hpi), harvest the cells using a cell scraper and prepare cell lysates for Western blot analyses.
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Materials
| Name | Company | Catalog Number | Comments |
| 4D-Nucleofector Core Unit (electroporation apparatus II) | Lonza (Basel, Switzerland) | AAF-1002B | |
| AB-Serum | Sigma Aldrich Chemie GmbH (Steinheim, Germany) | H4522 | Dendritic cell cultivation |
| ACD-A | Sigma-Aldrich Chemie GmbH (Steinheim, Germany) | 9007281 | |
| Amaxa P3 Primary Cell 4D-Nucleofector X Kit L (electroporation kit apparatus II) | Lonza (Basel, Switzerland) | V4XP-3024 | |
| Amersham ECL Prime Western Blotting Detection Reagent | GE Healthcare (Solingen, Germany) | RPN2232 | Western Blot Detection |
| Ammonium persulfate (APS) | Sigma Aldrich Chemie GmbH (Steinheim, Germany) | A3678 | |
| anti-mouse-IgG (mouse, polyclonal, HRP) | Cell Signaling (Leiden, Netherlands) | 7076 | Western Blot detection |
| anti-rabbit-IgG (goat, polyclonal, HRP) | Cell Signaling (Leiden, Netherlands) | 7074 | Western Blot detection |
| BD FACS Canto II Flow Cytometer | BD Biosciences (Heidelberg, Germany) | 338962 | |
| Blotting Chamber Fastblot B44 | Biometra (Göttingen, Germany) | 846-015-100 | |
| CCR7 (mouse, Pe-Cy7) | BioLegend (Fell, Germany) | 557648 | Flow cytometry Dilution: 1:100 Clone: G043H7 |
| CD11c (mouse, Pe-Cy5) | BD Biosciences (Heidelberg, Germany) | 561692 | Flow cytometry Dilution: 1:100 Clone: B-ly6 |
| CD14 (mouse, PE) | BD Biosciences (Heidelberg, Germany) | 555398 | Flow cytometry Dilution: 1:100 Clone: M5E2 |
| CD3 (mouse, FITC) | BD Biosciences (Heidelberg, Germany) | 555332 | Flow cytometry Dilution: 1:100 Clone: UCHT1 |
| CD80 (mouse, V450) | BD Biosciences (Heidelberg, Germany) | 560442 | Flow cytometry Dilution: 1:100 Clone: L307.4 |
| CD83 (mouse, APC) | eBioscience Thermo Fisher Scientific (Langenselbold, Germany) | 17-0839-41 | Flow cytometry Dilution: 1:200 Clone: HB15e |
| CD86 (mouse, PE) | BD Biosciences (Heidelberg, Germany) | 553692 | Flow cytometry Dilution: 1:100 |
| EVOS FL Cell Imaging | System AMG/Life Technologies (Carlsbad, USA) | AMF4300 | |
| FIP200 (rabbit) | Cell Signaling (Leiden, Netherlands) | 12436 | Western Blot detection Dilution: 1:1000 Clone: D10D11 |
| Gene Pulser II apparatus (electroporation apparatus I) | BioRad Laboratories GmbH (München, Germany) | 165-2112 | |
| GM-CSF (4x104 U/mL) | Miltenyi Biotec (Bergisch Gladbach, Germany) | 130-093-868 | |
| HLA-DR (mouse, APC-Cy7) | BioLegend (Fell, Germany) | 307618 | Flow cytometry Dilution: 1:200 Clone: L243 |
| HSV-1/17+/CMV-EGFP/UL43 | BioVex | DC infection | |
| IL-1β (0.1x106 U/mL) | Cell Genix GmbH (Freiburg, Germany) | 1411-050 | |
| IL-4 (1x106 U/mL) | Miltenyi Biotec (Bergisch Gladbach, Germany) | 130-093-924 | |
| IL-6 (1x106 U/mL) | Cell Genix GmbH (Freiburg, Germany) | 1404-050 | |
| ImageQuant LAS 4000 | GE Healthcare (Solingen, Germany) | 28955810 | |
| L-glutamine | Lonza (Basel, Switzerland) | 17-605E | |
| LIVE/DEAD Fixable Violet dead cell stain kit | Life Technologies (Carlsbad, CA, USA) | L34964 | L/D staining in Flow cytometry |
| Lymphoprep | Alere Technologies AS (Oslo, Norway) | 04-03-9391/01 | |
| Magnesium chloride | Carl Roth GmbH (Karlsruhe, Germany) | A537.1 | |
| Megafuge 2.0 RS | Heraeus (Hanau, Germany) | 75015505 | |
| Neubauer counting chamber | Brand (Wertheim, Germany) | 717805 | |
| Nunc Cell culture flasks (175.0 cm2) | Thermo Scientific (Rockford, USA) | 159910 | |
| Paraformaldehyde, 16 % | Alfa Aesar, Haverhill, USA | 43368.9M | |
| PerfectSpin 24 Plus | Peqlab (Erlangen, Germany) | C2500-R-PL | |
| PGE2 (1 mg/mL) | Pfizer (Berlin, Germany) | BE130681 | |
| Phosphate buffered saline (PBS) | Lonza (Basel, Switzerland) | 17-512F | |
| RestoreTM Western Blot Stripping Buffer | Thermo Scientific, Rockford, USA | 21059 | |
| Rocking Platform wt 15 | Biometra (Göttingen, Germany) | 042-590 | |
| RotiBlock | Carl Roth GmbH (Karlsruhe, Germany) | A151.4 | |
| Roti-Load 1 (4x) | Carl Roth GmbH (Karlsruhe, Germany) | K929.3 | |
| Rotiphorese Gel 30 (37.5:1) | Carl Roth GmbH (Karlsruhe, Germany) | 3029.1 | |
| RPMI 1640 | Lonza (Basel, Switzerland) | 12-167F | |
| Sodium dodecyl Sulfate (SDS) | Carl Roth GmbH (Karlsruhe, Germany) | 2326.2 | |
| Thermomixer comfort | Eppendorf (Hamburg, Germany) | 5355 000.011 | |
| TNF-α (10 μg/mL) | Peprotech (Hamburg, Germany) | 300-01A | |
| Tris | Carl Roth GmbH (Karlsruhe, Germany) | 4855.3 | |
| Trypan blue solution (0.4 %) | Sigma-Aldrich Chemie GmbH (Steinheim, Germany) | T8154 | |
| Tween 20 | Carl Roth GmbH (Karlsruhe, Germany) | 9127.1 | |
| Whatman 0.2 μm nitrocellulose membrane | GE Healthcare (Solingen, Germany) | 10600001 | |
| WhatmanTM Chromatography Paper 3 mm Chr | Fisher Scientific GmbH (Schwerte, Germany) | 3030917 |
