The Study of Host Immune Responses Elicited by the Model Murine Hookworms Nippostrongylus brasiliensis and Heligmosomoides polygyrus

T. Bouchery1, B. Volpe1, K. Shah1, L. Lebon1, K. Filbey2, G. LeGros2, N. Harris1

1 Global Health Institute, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, 2 Malaghan Institute of Medical Research, Wellington
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.34
Online Posting Date:  December, 2017
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Abstract

Hookworm infections (Necator americanus or Ancylostoma duodenale) represent a major neglected tropical disease, affecting approximately 700 million people worldwide, and can cause severe morbidity due to the need for these worms to feed on host blood. N. brasiliensis and H. polygrus, both rodent parasites, are the two most commonly employed laboratory models of experimental hookworm infection. Both parasites evoke type 2 immune responses, and their use has been instrumental in generating fundamental insight into the molecular mechanisms of type‐2 immunity and for understanding how the immune response can control parasite numbers. Here we provide a complete set of methods by which to investigate the natural progression of infection and the host immunological responses in the lung and intestine of H. polygyrus– and N. brasiliensis–infected mice. Detailed information is included about the most important parasitological and immunological measurements to perform at each time point. © 2017 by John Wiley & Sons, Inc.

Keywords: AAM; hookworms; H. polygyrus; ILC2s; N. brasiliensis; Th2

     
 
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Table of Contents

  • Introduction
  • Section 1: Infection and Assessment of Parasite Burden After Hookworm Infection
  • Basic Protocol 1: Enumeration of H. polygyrus Parasite Numbers Within Infected Animals
  • Support Protocol 1: Infection of Mice with H. polygyrus
  • Support Protocol 2: Infection of Mice with N. brasiliensis
  • Section 2: Assessment of Tissue Damage After Hookworm Infection
  • Basic Protocol 2: Quantitation of Alveolar Damage (Emphysema) Following N. brasiliensis Infection
  • Basic Protocol 3: Quantitation of Lung Hemorrhage Following N. brasiliensis Infection
  • Section 3: Assessment of the Type 2 Protective Immune Response Raised Against Hookworms
  • Basic Protocol 4: Analysis of Granuloma Formation and Composition Following H. polygyrus Infection
  • Support Protocol 3: Antigen Retrieval Methods
  • Basic Protocol 5: Evaluation of Antibody Response Elicited by H. polygyrus or N. brasiliensis Infection Using Parasite‐Specific Antibody ELISA
  • Basic Protocol 6: Characterization of the Type 2 Myeloid Cell Response Following Infection with N. brasiliensis or H. polygyrus
  • Basic Protocol 7: Characterization of the Type 2 Lymphoid Cell Response Following Infection with N. brasiliensis or H. polygyrus
  • Support Protocol 4: Isolation of Lung Immune Cells After Nb Infection
  • Support Protocol 5: Isolation of Immune Cells from the Small Intestinal Lamina Propria
  • Support Protocol 6: Isolation of Immune Cells from Intestinal Granulomas After Hp Re‐Infection
  • Support Protocol 7: Isolation of Leukocytes from the Peritoneal Cavity of Hp or Nb Infected Mice
  • Support Protocol 8: Isolation of Mediastinal Mesenteric Immune Cells
  • Basic Protocol 8: Imaging the Skin Invasion by Infective N. brasiliensis Larvae
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enumeration of H. polygyrus Parasite Numbers Within Infected Animals

  Materials
  • Hp‐infected mouse from 2 days post‐infection to 40 days post‐infection ( protocol 2)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS, Gibco, cat. no. 10010‐015)
  • Dissecting instruments including vessel scissors, pointed probe, and forceps
  • 12‐well plastic tissue culture dishes (Cellstar, cat. no. 665180)
  • Glass plates (approximately 10 × 10 cm)
  • Microscope slides
  • Stereomicroscope
  • 50‐ml conical polypropylene tubes (BD Falcon, cat. no. 352070)
  • Parafilm strips (cut to 5 mm × 3 cm)
  • 3‐ml transfer pipets
  • 4‐cm plastic Petri dish (TPP, cat. no. 93060) with a grid (approximately 0.8 × 0.8 cm) drawn on the back using a water‐resistant or diamond pen
  • Additional reagents and equipment for euthanasia of the mouse (Donovan & Brown, )

Support Protocol 1: Infection of Mice with H. polygyrus

  Materials
  • Infective third‐stage Hp larvae (iL3; the larvae are obtained from the maturation of eggs contained in the feces of an infective animal and isolation by Baermann technique as described in Camberis et al., )
  • Sterile Dulbecco's phosphate‐buffered saline (DPBS; Gibco, cat. no. 14040117)
  • Susceptible C57Bl/6 J mice (the CBA strain is also susceptible, while BALB/c and SJL strains are more resistant)
  • 50‐ml conical centrifuge tubes (BD Falcon, cat. no. 352070)
  • 4‐cm plastic Petri dish (TPP, cat. no. 93060) with a grid (approximately 0.8 × 0.8 cm) drawn on the back using a water‐resistant or diamond pen
  • Stereomicroscope
  • Gavage needle (5 cm in length)
  • 1‐ml disposable syringes, sterile

Support Protocol 2: Infection of Mice with N. brasiliensis

  Materials
  • Infective third‐stage Nb larvae (iL3; the larvae are obtained from the maturation of eggs contained in the feces of an infective animal; see Camberis et al., , for details)
  • Sterile Dulbecco's phosphate‐buffered saline (DPBS, Gibco, cat. no. 14040117)
  • C57Bl/6 J mice (most strains of mice are susceptible to Nb; however, FVB/N mice have been reported to be resistant)
  • 50‐ml conical centrifuge tubes (BD Falcon, cat. no. 352070)
  • 4‐cm plastic Petri dish (TPP, cat. no. 93060) with a grid (approximately 0.8 × 0.8 cm) drawn on the back using a water‐resistant or diamond pen
  • Stereomicroscope
  • 1‐ml disposable syringes, sterile
  • 25‐G needle (BD, cat. no. 300600)

Basic Protocol 2: Quantitation of Alveolar Damage (Emphysema) Following N. brasiliensis Infection

  Materials
  • Nb‐infected mice ( protocol 3; see Table 17.2.200 to chose appropriate time point of analysis)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS, Gibco, cat. no. 10010‐015)
  • 2% paraformaldehyde (PFA; see recipe for 4%)
  • Dissecting instruments including scissors and forceps
  • 25‐G needle (BD, cat. no. 300600)
  • 10‐ml syringe (Braun, cat. no. 4616103V)
  • 18‐G cannula (BD Insyte, cat. no. 381247)–remove the needle from inside the cannula
  • Yarn
  • 3‐ml syringe (Braun, cat. no. 2020‐10)
  • Catheter attached to perfusion controller (Dial‐a‐Flow; Hospira)
  • Ring stand
  • Automated tissue processor ASP200 (Leica)
  • Microtome: Microm Hyrax AAM5
  • Additional reagents and equipment for euthanasia of the mouse (Donovan & Brown, ) and immunohistochemical techniques including paraffin embedding, sectioning, and hematoxylin/eosin staining (Hofman & Taylor, )

Basic Protocol 3: Quantitation of Lung Hemorrhage Following N. brasiliensis Infection

  Materials
  • Nb‐infected mice (2 to 4 days post‐infection; see Camberis et al., ; also see comments in protocol 1)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010‐015), cooled on ice
  • RBC lysis buffer (Biolegend, cat. no. 420301)
  • Flow buffer (see recipe)
  • Live/dead aqua fixable viability dye (Thermo Fisher, cat. no. L34957)
  • Anti‐CD16/32 mAb, clone 24G2 (1 μg/ml)
  • Antibodies for flow cytometry (see Table 17.2.200)
  • Flow cytometry compensation beads (One Comp eBeads, Thermo Fisher, cat. no. 01‐1111‐42)
Table 7.2.3   MaterialsAntibody Mix for Neutrophil Detection in BAL

Antibody Dilution
CD45‐AF700 (Clone 30F11, Biolegend) 1:400
Ly6G‐AF647 (Clone 1A8, Biolegend) 1:200
Gr1‐PE (RB6‐8C5, eBioscience) 1:200
CD11b‐FITC (M1/70, Biolegend) 1:200
SiglecF‐PE‐CF594 (E50‐2440, BD) 1:200
Live dead aqua 1:250

  • Dissecting instruments including scissors and forceps
  • 18‐G cannula (BD Insyte, cat. no. 381247)–remove the needle from inside the cannula
  • Round‐bottom flow tubes, 5 ml (BD, cat. no. 352054) or 1.4 ml (Micronic, cat. no. MP32022)
  • Refrigerated centrifuge
  • 70‐μm filter gauze (cut small square of about 1 × 1 cm)
  • Flow cytometer with 4 to 5 lasers (LSRII or Fortessa, BD; access to a flow cytometer usually requires training; also see Robinson et al., )
  • Additional reagents and equipment for euthanasia of the mouse (Donovan & Brown, ), counting cells (Phelan & May, ), and flow cytometry (Robinson et al., )

Basic Protocol 4: Analysis of Granuloma Formation and Composition Following H. polygyrus Infection

  Materials
  • Hp‐infected mice ( protocol 2)
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010‐015)
  • 4% paraformaldehyde (PFA; see recipe)
  • 70% ethanol
  • Xylene
  • Ethanol: 100%, 96%, and 70% (v/v)
  • Antigen retriever (Lab Vision™ PT Module, Thermo Scientific) or proteinase K solution (Applichem, cat. no. A4392.0005)
  • Na‐citrate buffer (see recipe)
  • Blocking solution (see recipe)
  • Primary antibodies (Table 17.2.200)
  • Secondary antibodies of appropriate species (Table 17.2.200)
  • 1 mg/ml DAPI (Sigma, cat. no. D9542)
  • Mounting medium: ProLong Gold antifade reagent (Molecular Probes)
  • Transparent nail polish
Table 7.2.4   MaterialsAntibodies for Immunostaining of Hp Granulomas

Primary antibody Cell type Ag retrieval protocola Concentration Secondary antibody
F4/80 (Cl:A3‐1, Bio‐Rad) Macrophages PK/Na‐citrate pH 6.00 1:50 Anti‐rat (A11077, Life Technologies)
Arginase1 (LS‐B4789, LSBio) Macrophages Na‐citrate pH 6.00 1:50 Anti‐rabbit (A10042, Life Technologies)
MPO (ab9535, Abcam) Neutrophils/monocytes PK 1:50 Anti‐rabbit (A10042, Life Technologies)
Ly6C (ER‐MP20, Thermo Scientific) Monocytes PK 1:100 Anti‐rat (A11077, Life Technologies)

 aaAlso see Hofman & Taylor ( ). PK, proteinase K.
  • Scissors
  • Petri dish
  • Tissue paper (Kimwipes)
  • Forceps
  • 26‐G needle (BD, cat. no. 300600)
  • 6‐well plate or 50‐ml conical centrifuge tube (e.g., BD Falcon)
  • Swiss roll containers (PrintMate™ Slotted Cassettes, Thermo Scientific)
  • Hydrophobic pen (DAKO)
  • Immunostaining moisture chamber (H4262, ProSciTech)
  • Glass coverslips (Menzel‐Gläser, 24 × 50 mm)
  • Fluorescence microscope
  • Additional reagents and equipment for euthanasia of the mouse (Donovan & Brown, ), immunohistochemical techniques including paraffin embedding, sectioning, and hematoxylin/eosin staining (Hofman & Taylor, ), and antigen retrieval ( protocol 7; also see Hofman & Taylor, )

Support Protocol 3: Antigen Retrieval Methods

  Materials
  • Na‐Citrate buffer (see recipe)
  • Antigen retriever (Lab Vision™ PT Module, Thermo Scientific)
  • 20 μg/ml proteinase K (Sigma‐Aldrich) in PBS (Gibco, cat. no. 10010‐015)
  • 95°C heat block or water bath

Basic Protocol 5: Evaluation of Antibody Response Elicited by H. polygyrus or N. brasiliensis Infection Using Parasite‐Specific Antibody ELISA

  Materials
  • Excretory‐secretory (ES) products from L3 and/or L5 worms: detailed methods on how to prepare ES products from Nb can be found in Camberis et al. ( ) and for Hp in Johnston et al. ( ) and Valanparambil et al. ( ). In all cases, ES should be stored in small aliquots at –80°C and the necessary quantity thawed immediately prior to use.
  • ELISA coating buffer (see recipe)
  • ELISA washing buffer (see recipe)
  • Appropriate monoclonal antibodies for use as standards (see Table 17.2.200 for a list of antibodies).
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010‐015)
  • Bovine serum albumin (BSA; Sigma‐Aldrich, cat. no. A2153)
  • ELISA blocking buffer (see recipe)
  • Appropriate capture and detection monoclonal antibodies (see Table 17.2.200 for a list of antibodies)
  • 4‐nitrophenyl phosphate disodium salt hexahydrate (pNPP, Sigma‐Aldrich, cat. no. 71770) powder dissolved at 1 mg/ml in diethanolamine buffer
  • Diethanolamine buffer (see recipe)
  • Stop buffer (1 M NaOH; optional)
Table 7.2.5   MaterialsAntibody Anti‐ES Specific ELISA

Type of Ab studied ES coating Standard Samples Secondary Ab (use 1:1000)
H. polygyrus‐specific ELISA
IgG1 1 μg/ml Use 2nd infection serum; use range from 1:1000 to 1:2,000,000 Serum 1:30 for primary infection Serum 1:200 for secondary infection Southern Biotech, cat. no. 1070‐06
IgG2a/c a 1 μg/ml Use 2nd infection serum; use range from 1:3 to 1:6000 1:10 for all Southern Biotech, cat. no. 1080‐04
IgE 10 μg/ml Use 2nd infection serum; use range from 1:100 to 1:200,000 1:100 for all Southern Biotech, cat. no. 1130‐04
IgM 5 μg/ml Use 2nd infection serum; use range from 1:10 to 1:20,000 1:100 for all Southern Biotech, cat. no. 1021‐04
N. brasiliensis‐specific ELISA
IgE 5 μg/ml Use 3rd infection serum; use range from 1:10 to 1:20,000 1:10 for serum Clone 6HD5
IgG1 5 μg/ml Use 3rd infection serum; use range from 1:10 to 1:20,000 1:10 for all Southern Biotech, cat. no. 1070‐06
IgG2a/c 5 μg/ml Use 3rd infection serum; use range from 1:10 to 1:20,000 1:10 for all Southern Biotech, cat. no. 1080‐04

 aAnti‐IgG2a is cross‐reactive for IgG2c. C57BL/6 mice, contrary to BALB/c mice, have no IgG2a (Esser‐von Bieren, Volpe, Kulagin, et al., 2015).
  • 96‐well flat‐bottom ELISA plates (Nunc MaxiSorp 442404).
  • 96‐well round‐bottom plate
  • Spectrophotometer (e.g., Benchmark Plus from Bio‐Rad)
  • Software to analyze results: ELISA reader software, GraphPad Prism, or Microsoft Excel

Basic Protocol 6: Characterization of the Type 2 Myeloid Cell Response Following Infection with N. brasiliensis or H. polygyrus

  Materials
  • Immune cells isolated from the relevant tissue of Nb or Hp infected mice (see Tables 17.2.200 and 17.2.200 for relevant tissues and timepoints and Support Protocols protocol 114 to protocol 158 for cell isolation procedures)
  • Flow buffer (see recipe)Live/dead aqua fixable viability dye (Thermo Fisher, cat. no. L34957)
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010‐015)
  • 70% ethanol1.5% paraformaldehyde (PFA; see recipe for 4%)
  • Cold methanol
  • Flow cytometry compensation beads (One Comp eBeads; Thermo Fisher, cat. no. 01‐1111‐42)
  • Round‐bottom flow tubes: 5 ml (BD, cat. no. 352054) or 1.4 ml (Micronic, cat. no. MP32022)
  • Refrigerated centrifuge
  • 70‐µm gauze
  • Flow cytometer (BD Fortessa, BD LSR or equivalent machine)
  • Additional reagents and equipment for flow cytometry (Robinson et al., )

Basic Protocol 7: Characterization of the Type 2 Lymphoid Cell Response Following Infection with N. brasiliensis or H. polygyrus

  Materials
  • Immune cells isolated from the relevant tissue of Nb‐ or Hp‐infected mice (see Tables 17.2.200 and 17.2.200 for relevant tissues and time points and Support Protocols protocol 114 to protocol 158 for cell isolation protocols)
  • Flow buffer (see recipe)
  • Live/dead aqua fixable viability dye (Thermo Fisher, cat. no. L34957)
  • Anti‐CD16/32 (24G2) and other antibodies (see Table 17.2.200)
  • Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher, cat. no. 00‐5523‐00)
  • Flow cytometry compensation beads (One Comp eBeads; Thermo Fisher, cat. no. 01‐1111‐42)
Table 7.2.7   MaterialsAntibody Mix for Flow Cytometric Analysis of Innate Lymphoid Cells

Antibody Dilution
CD45‐AF700 (Clone 30F11, Biolegend) Surface mix 1:400
CD25‐PECy7 (Clone PC61, Biolegend) Surface mix 1:100
CD4‐APC (Clone GK1.5, Biolegend) Surface mix 1:200
SiglecF‐PE (Clone E50‐2440, BD) Surface mix 1:200
Gr1‐PE (CloneRB6‐8C5, eBioscience) Surface mix 1:50
B220‐PE (Clone RA3‐6B2, eBioscience) Surface mix 1:100
CD11b‐PE (Clone M1/70, eBioscience) Surface mix 1:400
CD11c‐PE (Clone N418, Biolegend) Surface mix 1:400
NKp46‐PE (Clone 29A1.4, Biolegend) Surface mix 1:100
CD8‐PE (Clone 53‐6.7, eBioscience) Surface mix 1:400
F4/80‐APC‐eFluor780 (clone BM8, eBiosicience) Surface mix 1:200
KLRG1‐BV421 (Clone 2F1, BD) Surface mix 1:200
CD127‐Biotin (Clone A7R34, Biolegend) Surface mix 1:200
Gata‐3‐PerCp‐eF710 (Clone TWAJ,eBioscience) Intracellular mix in perm buffer 1:50
Streptavidin‐FITC (Biolegend) In flow buffer 1:1000

  • Round‐bottom flow tubes: 5 ml (BD, cat. no. 352054) or 1.4 ml (Micronic, cat. no. MP32022)
  • Refrigerated centrifuge
  • Flow cytometer
  • 70‐μm gauze
  • Additional reagents and equipment for flow cytometry (Robinson et al., )

Support Protocol 4: Isolation of Lung Immune Cells After Nb Infection

  Materials
  • Nb‐infected mice (see Table 17.2.200).
  • IMDM medium containing 10% fetal bovine serum (FBS)
  • Lung digestion buffer (see recipe)
  • Flow buffer (see recipe)
  • RBC lysis buffer (Biolegend, cat. no. 420301)
  • 24‐well tissue culture plates (Cell Star, cat. no. 662160)
  • 6‐well plates (Cell Star, cat. no. 657160)
  • Dissecting instruments including probe, pointed vessel scissors, and forceps
  • Orbital shaker (with temperature control)
  • 3‐ml syringe (Braun, cat. no. 2020‐10)
  • 18‐G needle (BD, cat. no. 305196)
  • 70‐μm cell strainer (Fisher, cat. no. 22363548)
  • 50‐ml tubes (BD, cat. no. 352070)
  • Plunger of 2‐ml syringe
  • Additional reagents and equipment for euthanasia of mice (Donovan & Brown, ) and counting viable cells by trypan blue exclusion (Phelan & May, )

Support Protocol 5: Isolation of Immune Cells from the Small Intestinal Lamina Propria

  Materials
  • Infected mice
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010‐015)
  • 70% ethanol
  • Intestinal digestion solution numbers #1 to 5 (see reciperecipes)
  • Dissecting instruments including probe pointed vessel scissors and forceps
  • Petri dish
  • Gavage needle
  • 10‐ml syringes (Braun. cat. no. 201803)
  • Tissue strainer (e.g., standard steel tea strainer)
  • Waste bottle
  • Petri dish, 10 cm
  • 50‐ml conical centrifuge tubes (e.g., BD, cat. no. 352070)
  • Plastic weighing boats
  • Orbital shaker (with temperature control)5‐ml syringes
  • 18‐G needle (BD, cat. no. 305196)
  • 70‐μm cell strainers (Fisher, cat. no. 22363548)
  • Plunger from 2‐ml syringe
  • Refrigerated centrifuge
  • 40‐μm cell strainers (Fisher, cat. no. 22363547)
  • Additional reagents and equipment for euthanasia of mice (Donovan & Brown, ) and counting viable cells by trypan blue exclusion (Phelan & May, )

Support Protocol 6: Isolation of Immune Cells from Intestinal Granulomas After Hp Re‐Infection

  Materials
  • Infected mice
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010‐015)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010‐015), cooled on ice
  • 0.5% bovine serum albumin (BSA; Sigma‐Aldrich, cat. no. A2153) in PBS
  • Granuloma digestion medium (see recipe)
  • IMDM medium containing 10% fetal bovine serum (FBS)
  • Flow buffer (see recipe)
  • RBC lysis buffer (Biolegend, cat. no. 420301)
  • Dissecting instruments including pointed probe vessel scissors and forceps
  • Petri dish
  • 24‐well plate
  • Glass plate
  • Biopsy punch, 1.5 mm (Miltex, cat. no. 33‐31A)
  • Orbital shaker
  • 70‐μm cell strainers (Fisher, cat. no. 22363548)
  • 50‐ml conical centrifuge tubes (e.g., BD, cat. no. 352070)
  • Plunger from 2‐ml syringe
  • Refrigerated centrifuge
  • Additional reagents and equipment for euthanasia of mice (Donovan & Brown, ) and counting viable cells by trypan blue exclusion (Phelan & May, )

Support Protocol 7: Isolation of Leukocytes from the Peritoneal Cavity of Hp or Nb Infected Mice

  Materials
  • Mouse
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010‐015), cooled on ice
  • Dissecting instruments including probe pointed vessel scissors and forceps
  • 25‐G needle (BD, cat. no. 300600)
  • 15‐ml conical tubes (Corning Falcon, cat. no. 352095)
  • Additional reagents and equipment for euthanasia of mice (Donovan & Brown, )

Support Protocol 8: Isolation of Mediastinal Mesenteric Immune Cells

  Materials
  • Mouse
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010‐015), cooled on ice
  • Flow buffer (see recipe)
  • Dissecting instruments including probe pointed vessel scissors and forceps
  • 70‐μm cell strainers (Fisher, cat. no. 22363548)
  • 50 ml conical tubes (BD, cat. no. 352070)
  • Plunger from 1‐ml syringe (Braun, cat. no. 2020‐03)
  • Refrigerated centrifuge
  • Additional reagents and equipment for euthanasia of mice (Donovan & Brown, )

Basic Protocol 8: Imaging the Skin Invasion by Infective N. brasiliensis Larvae

  Materials
  • Infective third‐stage Nb larvae (iL3; the larvae are obtained from the maturation of eggs contained in the feces of infective animals, see Camberis et al., , for details of procedure)
  • CFSE (see recipe)
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010‐015), cooled on ice
  • Wash solutions I and II for L3 staining (see reciperecipes)
  • Culture solution for L3 staining (see recipe)
  • 1.5‐ml tubes (Sarstedt, cat. no. 72.706), covered with tin foil
  • 24‐well culture plate (Cell Star, cat. no. 662160)
  • 15‐ml conical tubes (Corning Falcon, cat. no. 352095)
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Figures

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Literature Cited

Literature Cited
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