Quantitative Analysis of Plasmodium berghei Liver Stages by

429

Robert Ménard (ed.), Malaria: Methods and Protocols, Methods in Molecular Biology, vol. 923,

DOI 10.1007/978-1-62703-026-7_30, © Springer Science+Business Media, LLC 2013

Chapter 30

Quantitative Analysis of Plasmodium berghei Liver Stages

by Bioluminescence Imaging

Takeshi Annoura , Séverine Chevalley , Chris J. Janse ,

Blandine Franke-Fayard , and Shahid M. Khan

Abstract

We describe simple and sensitive in vitro and in vivo assays to analyze Plasmodium liver stage development

using transgenic P. berghei parasites ( Pb GFP-Luc

con

), which express the bioluminescent reporter protein,

luciferase. In these assays, parasite development in hepatocytes is visualized and quanti ﬁ ed by real-time

bioluminescence imaging both in culture and in live mice. We also describe quanti ﬁ cation of in vitro liver-

stage development by measuring luminescence using a microplate reader. Reporter-parasite based

quanti ﬁ cation of liver-stage development is faster and correlates very well with established quantitative

RT-PCR methods currently used to assess parasite development inside hepatocytes, both in live mice and

in culture.

Key words: Malaria , Plasmodium berghei , Sporozoites , Preerythrocytic stages , Liver , Hepatocytes ,

Luciferase , Luminescence , In vivo imaging , Drug screening , Mice

Quantitative analysis of Plasmodium liver-stage development both

in vivo in laboratory rodents and in vitro in cultured liver cells is

hampered by the low levels of parasite infection and by the compli-

cated methods required to monitor parasite growth. Currently,

one of the standard ways to assess drug ef ﬁ cacy against liver stages

is to monitor liver stage development, both in vitro and in vivo, by

quantitative RT-PCR (qRT-PCR) methods (

1– 5 ) , and this is both

time-consuming and expensive. Other studies have involved assess-

ing parasite viability and direct quanti ﬁ cation of development by

microscopy (

6 ) , RNA hybridization ( 7 ) , or infrared ﬂ uorescence

1. Introduction

430 T. Annoura et al.

scanning system ( 8 ) . However, these methods not only are prone

to large variations between observers but are also time-consuming

given the very low infection rates (generally less than 2%) observed

in cultured hepatocytes (

8 ) . Here we describe simple and sensitive

in vitro and in vivo assays to visualize and quantify liver-stage devel-

opment using the transgenic P. berghei parasites Pb GFP-Luc

con

which expresses the bioluminescent reporter protein, luciferase.

The luminescence-based quanti ﬁ cation of parasite development in

hepatocytes has been shown to correlate very well with established

quantitative RT-PCR methods (

9 ) . Speci ﬁ cally, analysis of liver

infections by whole-body real-time imaging correlates well with

quantitative RT-PCR analysis of extracted livers. In addition lumi-

nescence-based quanti ﬁ cation of liver stage parasites in cultured

hepatocytes by real-time imaging or using a microplate reader also

correlates well with quantitative RT-PCR methods. Both the

in vitro and in vivo liver imaging assays are amenable to screen

inhibitors and vaccines against liver stages (

9, 10 ) . Real-time imag-

ing of liver stages in mice has been successfully used to examine

host factors regulating liver infections and to monitor liver-stage

development of genetically attenuated parasites (

11 ) . Importantly,

the in vivo imaging assays allow the course of an infection to be

monitored, both throughout liver-stage parasite development and

in the blood stage of infection without sacri ﬁ cing the animal, and

therefore, can greatly reduce the number of experimental animals

required to determine drug sensitivity. The simplicity and speed of

quantitative analysis of liver-stage development by real-time imag-

ing compared to the PCR-based methodologies, as well as the pos-

sibility to analyze parasite development in live mice without surgery,

should greatly enhance and simplify analyzing the effect of drugs

and vaccines on the liver stage of Plasmodium .

For the assays the reporter parasite line Pb GFP-Luc

con

(676m1cl1)

is used, which expresses a fusion protein of GFP (mutant3) and

ﬁ re ﬂ y luciferase (LUC-IAV) under the control of the constitutive

eef1 a promoter (

12 ) . For details of Pb GFP-Luc

con

, see RMgm-29

(

http://www.p berghei .eu/index.php?rmgm=29 ).

In our laboratory, mice of the following two strains are routinely

used: Swiss (OF1 ico, Construct 242, aged 6 weeks, 25–26 g) and

C57BL/6 (C57Bl/6Jico OF1, Construct 1, aged 6 weeks,

20–25 g). The mice are obtained from Charles River. Other (strains

or transgenic) mice can also be used for in vivo imaging experi-

ments (see Note 1).

2. Materials

2.1. Reporter Parasite

2.2. Laboratory

Animals

43130 Quantitative Analysis of Plasmodium berghei Liver Stages…

1. Sporozoites of parasite line Pb GFP-Luc

con

. This protocol

requires the collection of (large numbers) of sporozoites.

Sporozoites are removed from the salivary glands of infected

Anopheles stephensi mosquitoes at days 20–28 after feeding on

mice infected with Pb GFP-Luc

con

parasites. For procedures of

maintenance/rearing of mosquitoes and infection of mosqui-

toes, we refer to ref.

(13) .

2. Hepatocytes. The human hepatocyte carcinoma cell line Huh7

(JCRB0403, JCRB Cell Bank, JP) is used for in vitro cultures

of the liver stages.

3. Fetal bovine serum, heat-inactivated (FBS; Invitrogen; cat. no.

10108-165). Store at −20°C.

4. Phosphate-buffered saline (PBS). PBS stock solution (10×):

0.01 M KH

, 0.1 M Na

HPO

, 1.37 M NaCl, 0.027 M

KCl, pH 7.4. For a working solution, dilute the stock solution

with 9 volumes of distilled water, adjust the pH to 7.2 with

1.0 M HCl and sterilize by autoclaving for 20 min at 120°C.

5. Complete RPMI1640 culture medium. RPMI1640 medium

(Invitrogen; cat. no. 31870-025) supplemented with FBS to a

ﬁ nal concentration of 10 or 20% (v/v), 1% GlutaMAX

(Invitrogen; cat. no. 35050) and 1% penicillin–streptomycin

(MP Bio; cat. no. 1670049).

6. Trypsin, 0.05% (1×) with EDTA 4Na (Invitrogen; cat. no.

25300-054).

7. Cell culture lysis reagent (CCLR): Luciferase Assay System kit

(Promega, cat. no. E1500). For working solution, dilute the

“Cell Culture Lysis 5× Reagent” provided in the kit with

Milli-Q water.

8. Luciferase assay substrate solution: Luciferase Assay System Kit

(Promega, cat. no. E1500). For working solution, mix 1 vial of

luciferase assay substrate and 1 vial of 10 ml luciferase assay

buffer together. The mixed solution can be stored at −20°C

and can be subsequently freeze/thawed multiple times with-

out a signi ﬁ cant loss of activity, however, the solution must be

kept in the dark at all times.

D -Luciferin sodium salt. Dissolve 1 g D -luciferin in 12.5 ml

PBS to give a stock solution of 80 mg/ml and store at −20°C

in the dark in 500- m l aliquots. Thaw the stock solution prior to

use and inject into a mouse at a concentration of 120 mg/kg

body weight (i.e., 30 m l for a mouse of 20 g).

10. Inhibitors/antimalarial drugs: dissolve the powders in DMSO,

sterile Milli-Q water or culture medium in high concentration

as stock solution. Store at 4°C or −20°C. For serial dilutions,

dilute the working stock solutions with DMSO and/or culture

medium (see Note 2).

2.3. Reagents

432 T. Annoura et al.

1. Stereomicroscope (Leica M80) for mosquito dissection. Most

stereomicroscopes are suitable.

2. (Upright) light microscope (Leica DM2500 or Carl Zeiss

Standard 25 Zeiss) for counting sporozoites. All light micro-

scopes with 40× objective are suitable.

3. Inverted microscope, Leica DMIL for analysis of Huh7 cell

cultures. All inverted microscopes with 20× objective are

suitable.

4. Carbon dioxide gas source (for anesthesia of mosquitoes).

5. Precision forceps (Original Swiss Dumont precision forceps,

cat. no. K342.1). Most thin precision forceps are suitable.

6. Insulin needle syringe (BD Micro-Fine + U-100 insulin,

0.33 mm; 30 G × 8 mm, BD Medical, France). Most thin small

needle syringes are suitable.

7. Incubation (moist) chamber (COSMO BIO CO., LTD. cat.

no. KMB-10CG). All moist chambers are suitable.

8. Bürker-Türk counting chamber (Carl Roth GmbH, cat. no.

T730.1).

9. Tissue grinder of Polypropylene Pestle for 1.5-ml tubes (Carl

Roth GmbH, cat. no. P987.1).

10. 75-cm

cell culture ﬂ ask (Corning cell culture ﬂ asks; cat. no.

CLS3276).

11. CO

incubator (Thermo/Forma Scienti ﬁ c CO

Water Jacketed

Incubators, Model 3121). All CO

incubators for cell culture

are suitable.

12. Luminescence microplate reader: Wallac Multilabel Counter

1420 (PerkinElmer, NL). Other microplate readers that can

measure bioluminescence are suitable.

13. 24-well (Corning; cat. no. CLS3524).

14. 96-well optical ﬂ at-bottomed and black-framed microplates

(Nalge Nunc Intl.). All 96-well microplates with black frames

and clear ﬂ at bottoms are suitable for luminescence measure-

ment (see Note 3).

15. Vortex shaker (Ika Labortechnik).

16. Infra-red heat lamp (home-made or from Science Products).

17. Biohazard Class II safety cabinet (see Note 4).

18. Table-top centrifuge (Beckman Coulter Allegra, GS-6 centri-

fuge). Most table-top centrifuges with a swing-out rotor are

suitable but these need to have a carrier assembly for plates

(Beckman Coulter; MicroPlus Carrier Assembly for GH-3.8

Rotors cat. no. BK362394).

19. Contura HS-40 shaver (Wella), used for removal of the fur

from the skin of mice by shaving (Optional).

2.4. Equipment

43330 Quantitative Analysis of Plasmodium berghei Liver Stages…

20. Anesthesia system (i.e., XGI-8 gas connected to the Lumina II

from Caliper) for anesthesia of mice prior to and during in vivo

imaging. Mice are anesthetized in the “induction chamber,”

which is pre- ﬁ lled with the anesthetic vapor (iso ﬂ uorane/air)

via the vaporizer unit, and are kept under anesthesia in the

imaging chamber by holding their muzzles close to a small

mask connected to the main vaporizer unit.

21. IVIS Lumina II System (Caliper Life Sciences, USA). All

in vivo imaging system with bioluminescent imaging option

are suitable for this purpose.

1. Imaging data are analyzed with the software provided with the

in vivo imaging system (i.e., LIVING IMAGE 4.1 for the

Lumina II from Caliper).

2. Microsoft Excel is used to conduct preliminary data analyses.

3. GraphPad Prism software (Graph-Pad software, Inc., USA) is

used for statistical analyses (best- ﬁ t) effective concentration

(EC50) calculation.

This protocol requires the collection of Pb GFP-Luc

con

sporozo-

ites from infected A. stephensi mosquitoes. Sporozoites are

obtained from dissected salivary glands at days 20–28 after blood

feeding. Maintenance/rearing and infection of mosquitoes are

performed as described in (

13 ) . The total number of mosquitoes

required is dependent on the experiment and the number of sali-

vary gland sporozoites per dissected mosquito; in our laboratory

we usually obtain sporozoites loads of 0.5–1 × 10

per mosquito

(see Fig.

1 for a schematic representation of the work ﬂ ow involv-

ing collection of sporozoites, in vitro culture and analysis of the

liver stages).

1. Transfer infected mosquitoes into a tissue net covered 50-ml

centrifuge tube.

2. Anesthetize mosquitoes with carbon dioxide (blown directly

into the tube). Keep the tube containing the anesthetized mos-

quitoes on ice until dissection.

3. Place the mosquito in one drop of PBS on a glass slide under a

stereomicroscope (magni ﬁ cation 5–20×).

4. Carefully remove the mosquito head from thorax using a for-

ceps and an insulin needle. In general the two glands, each

with three lobes, will remain attached to the head (see Fig.

2 ),

2.5. Software

3. Methods

3.1. Analysis

and Quanti ﬁ cation

of In Vitro Liver-Stage

Development

3.1.1. Collection

of Sporozoites

434 T. Annoura et al.

Fig. 1. Work ﬂ ow scheme for quantitative analysis of Plasmodium liver stages. Left hand side (LHS): liver-stage development

in vitro analyzed by measuring luminescence using a plate reader. Right hand side (RHS): liver-stage development in vitro

analyzed by real-time bioluminescence imaging of live mice.

Fig. 2. ( a ) The view of a mosquito’s head attached to six intact salivary gland lobes ( grey arrows ; stereomicroscope; ..

magni ﬁ cation). ( b ) GFP-expressing sporozoites of parasite line a Pb

GFP-Luc

con

in lobes of a salivary gland ( white arrows )

and free sporozoites (SPZ; ﬂ uorescence microscope).

43530 Quantitative Analysis of Plasmodium berghei Liver Stages…

but sometimes they remain within the thorax. In this case,

gently press the forceps on the thorax in order to release the

salivary glands.

5. Collect and transfer the two salivary glands (with six intact

lobes) by forceps into a 1.5-ml Eppendorf tube containing

100 m l of incomplete RPMI1640 medium. It is important to

pick only the salivary glands without contamination with other

mosquito organs.

6. Repeat steps 4–6 until the required number of salivary glands

is collected.

7. In order to obtain free sporozoites from the salivary glands,

disrupt the collected salivary glands using a PP-pestle homog-

enizer and Vortex shaker (see Note 5).

8. In order to determine the total number of collected sporozo-

ites, take a 10 m l-sample (from the 100 m l) and place in a

Bürker-Türk cell counter.

9. First allow the sporozoites to settle before counting the

sporozoites by placing the cell counter for 20 min in a moist

chamber.

10. Sporozoite suspensions.

(a) For the in vitro assays, dilute the sporozoite suspension with

complete RPMI1640 medium (10% FBS), to a ﬁ nal con-

centration of 0.3–10 × 10

sporozoites per 100 m l for trans-

fer to the Huh7 cultures (see Subheading

3.1.3 , step 2).

(b) For the in vivo assays, dilute the sporozoite suspension

with incomplete RPMI1640 medium to a ﬁ nal concentra-

tion 1–200 × 10

sporozoites per 50–800 m l for injection

into mice (see Subheading

3.2.2 , step 4).

For principles of Huh7 cell culture, such as storage, thawing and

maintenance of cells, see the Japanese Collection of Research

Bioresources (JCRB) (

http://huh7.com/huh7_cell_culture.html ).

Most manipulations for the in vitro cultivation of Huh7 cells (in

combination with P. berghei parasites) are performed in a Class II

safety cabinet.

1. Defrost frozen stock-solution of 1 ml Huh7 cells (1 × 10

cells

in 1 ml of complete RPMI1640 medium, containing 10%

DMSO) in 10 ml Cell complete RPMI1640 culture medium

(20% FBS) into a 50-ml centrifuge tube.

2. Centrifuge at 13,000 × g for 10 min at RT using a tabletop

centrifuge and remove the supernatant.

3. Resuspend the cells by adding 30 ml of complete RPMI1640

culture medium (20% FBS) and seed into 75-cm

tissue culture

ﬂ asks.

3.1.2. Culture of Huh7

Cells

436 T. Annoura et al.

4. Incubate the ﬂ asks for 12–24 h in a CO

incubator (37°C,

5% CO

5. After this culture period, eliminate dead cells by removing

culture medium and washing the plates with 20 ml PBS. Add

30 ml complete RPMI1640 medium (20% FBS) and incubate

the plates for several days (see Note 6).

6. For collecting Huh7 cells for the “sporozoite assays,” remove

the culture medium from the plates and “detach” the Huh7

cells by adding 3 ml of the trypsin–EDTA 4Na solution for a

period of 5–10 min at 37°C.

7. To the (detached) cell suspension add 17 ml complete

RPMI1640 medium (10% FBS) and transfer to 50-ml tubes.

8. Spin at 200 × g for 10 min at RT in a table-top centrifuge,

remove the supernatant, and resuspend the cells in 10 ml com-

plete RPMI1640 medium (10% FBS).

9. Collect a sample of 10 m l for determination of the numbers of

Huh7 cells by counting in a Bürker-Türk counting chamber.

10. Adjust the volume of the Huh7 cell suspension with complete

RPMI1640 medium to obtain a ﬁ nal concentration of 5 × 10

cells per ml.

11. Seed 5 × 10

Huh7 cells by adding 1 ml of the ﬁ nal Huh7-cell

suspension to wells of a 24-well plate.

12. Before adding sporozoites to the wells, incubate the 24-well

plates for 12–24 h in a CO

incubator (37°C; 5% CO

). Huh7

cells will cover 40–60% of the well.

1. Remove the culture medium from the wells of the 24-well

plates (see Subheading

3.1.2 , steps 12–13) and subse-

quently add 900 m l fresh, pre-warmed (37°C) complete

RPMI1640 medium (10% FBS). See Note 7 for additional

information about the timing of adding inhibitors/drugs

to the cultures.

2. Add 100 m l of the sporozoite suspension containing 0.3–10 × 10

sporozoites (see Subheading

3.1.1 , step 10) to each well.

3. Spin at 13,000 × g for 5 min at RT (table-top centrifuge;

Beckman Coulter Allegra, GS-6 centrifuge with carrier assem-

bly for plates swing unit).

4. Incubate at 37°C and 5% CO

for 2–3 h.

5. Remove free sporozoites from the wells by removing the

medium and replacing it with 1 ml of fresh pre-warmed com-

plete RPMI1640 medium. See Note 7 for additional informa-

tion about the timing of adding inhibitors/drugs to the

cultures.

6. Return plates to the incubator at 37°C and 5% CO

3.1.3. Sporozoite Invasion

and Culture of the Liver

Stages in Huh7 Cells (with

or Without Addition of

Inhibitors)

43730 Quantitative Analysis of Plasmodium berghei Liver Stages…

Parasite liver-stage development in Huh7 hepatocytes is analyzed

over a 52-h period after the addition of sporozoites. After sporozo-

ite invasion, most P. berghei parasites develop in 60–64 h into

mature liver schizonts. After 64 h, merozoites are released from

the hepatocyte as merosomes, packets of 100–200 merozoites sur-

rounded by host cell membrane. Therefore, liver-stage develop-

ment can be quanti ﬁ ed by measuring bioluminescence of lysed

cultured cells up to 52 h after sporozoite invasion, using a microplate

reader or by measuring bioluminescence directly in culture plates

using the IVIS Lumina II system (see Note 8). Usually, experi-

ments are performed in triplicate (3 culture wells per condition or

time-point).

1. Remove the culture medium from the wells of the 24-well

plates and add 1 ml PBS for washing.

2. Remove PBS and add 100 m l of 1× cell culture lysis reagent

(CCLR).

3. Mix the cells and the CCLR by pipetting until all cells are lysed

(when the bottom of the wells become clear and the lysis solu-

tion is homogenous).

4. Collect the cell lysis solution and transfer lysed cells from each

well to 1.5-ml Eppendorf tubes. These samples can be stored

at −80°C until ready to perform the luciferase assay.

5. When all samples have been collected and are ready for the

luminescence assay, add 100 m l of “luciferase assay substrate

solution” and 10 m l of the lysed cell samples into wells of a

black-framed 96-well plate. Samples containing uninfected

Huh7 cells are used as negative controls.

6. Measure the light reaction of each well for 10 s using a

microplate luminometer. The luciferase activities are expressed

as relative luminescence units (RLU) for each sample.

7. Export the data and proceed with statistical analysis of the

data.

This protocol requires Pb GFP-Luc

con

sporozoites collected from

salivary glands of infected A. stephensi mosquitoes at days 20–28

after blood feeding. Maintenance, rearing and infection of mosqui-

toes are performed as described in (

13 ) . The total number of mos-

quitoes required is dependent on the experiment and the number

of salivary gland sporozoites per dissected mosquito; in our labora-

tory we usually obtain sporozoites loads of 0.5–1 × 10

per mos-

quito (see Fig.

1 for a schematic representation of the work ﬂ ow

involving collection of sporozoites, infection of mice with sporo-

zoites and analysis of liver stage development).

3.1.4. Quanti ﬁ cation

of In Vitro Liver-Stage

Development (with or

Without Addition of

Inhibitors)

3.2. Analysis

and Quanti ﬁ cation

of In Vivo Liver-Stage

Development

438 T. Annoura et al.

Sporozoites of the Pb GFP-Luc

con

line are obtained from dissected

salivary glands from infected mosquitoes as described in

Subheading

3.1.1 .

1. Place the mice under an IR heat lamp 5–10 min before injec-

tion of the sporozoites (see step 3). The tail veins swell at the

higher temperature, simplifying the intravenous injection pro-

cedure. In addition, prepare anesthesia system in suf ﬁ cient

time, such as ﬁ lling the “induction chamber” with the anes-

thetic vapor (iso ﬂ uorane/air), to be able to inject the sporozo-

ites immediately after the puri ﬁ cation procedure.

2. Prepare the sporozoite suspensions as described in

Subheading

3.1.1 , step 10.

3. Dilute the sporozoite suspension with incomplete RPMI1640

medium, to a ﬁ nal concentration of 1–200 × 10

sporozoites

per 200 m l for injection into mice. This sporozoite suspension

is injected intravenously into the tail vein (see Note 9).

1. Prepare the in vivo imaging system for imaging the mice

(Fig.

3a shows the Lumina II and anesthesia system from

Caliper) (see Note 10).

2. For imaging liver stages, anesthetize infected mice at different

time points after sporozoite inoculation (e.g., 24, 40, 48 or

64 h) using the iso ﬂ uorane-anesthesia system.

3. Remove the fur from the ventral part of the body by shaving

using a Contura HS-40 shaver. Removal of the fur is performed

to prevent quenching of the light signal, and must be done

carefully as a hematoma might in ﬂ uence the imaging.

4. Inject 30 m l

D -luciferin substrate solution subcutaneously into

the neck of the anesthetized mouse (see Note 11).

5. Place the mouse on a piece of Art again paper and position it

under the camera in the centre of the sample stage (if needed,

ﬁ x with black tape). The gated sample stage is pre-warmed to

37°C and thereby stabilizes the body temperature of the mouse

(see Note 12).

6. Wait for 3 min before acquiring the bioluminescence image.

This period allows circulation of the D- luciferin substrate

within the body of the mouse.

7. Acquire the bioluminescent image. The bioluminescent signal

collected is linearly related to the exposure time within a range

of 5 s to 10 min. Routinely, we image infected mice for

60–180 s when mice have been injected intravenously with

1 × 10

sporozoites. If necessary, acquire a new image with

shorter or longer exposure time.

3.2.1. Preparation of

Sporozoites

3.2.2. Infection of Mice

with Sporozoites

3.2.3. Quanti ﬁ cation of

Liver-Stage Development

In Vivo (with or Without

Drug Treatment of Mice)

43930 Quantitative Analysis of Plasmodium berghei Liver Stages…

8. After exposure is complete, the overlay of the photographic

and luminescent picture is displayed. See Fig.

3b for represen-

tative images of luminescent signals of liver stages in mice

infected with Pb GFP-Luc

con

sporozoites at different time points

after infection.

9. Save imaging data for post-processing analysis (i.e., measure-

ment of the intensity of bioluminescent signals in a speci ﬁ c

area (see Subheading

3.2.4 )).

10. Remove the mouse from the imaging chamber and repeat steps

3–8 for a new mouse.

Fig. 3. ( a ) As an example, the in vivo imaging system “IVIS Lumina II” from Caliper is shown, which has been used in our

laboratory for real-time imaging of P. berghei liver stages in whole bodies of live mice and in isolated, non ﬁ xed livers.

( b ) In vivo images of the same mouse with a developing liver-stage infection, at different time points after intravenous

inoculation of 10,000 Pb GFP-Luc

con

sporozoites. At 64 h, merozoites are released from the liver into the blood circulation.

The relative luminescence units (RLU), at each time point, are shown under the picture of the mouse.

440 T. Annoura et al.

The whole-body bioluminescence images of mice provide a

qualitative assessment of the load/intensity of Pb GFP-Luc

con

liver

stages within an animal and this liver load can be directly compared

between different animals if the same measurement settings during

the experiments are maintained. Most in vivo imaging systems

software contain tool options that enable the quanti ﬁ cation of bio-

luminescent signal emanating from speci ﬁ c areas of the mouse

(“region of interest,” ROI).

1. Select the image to analyze.

2. Create region of interest (ROI; Liver) on the image (see

Fig.

3b ).

3. Determine the bioluminescent intensity in ROI. The measure-

ment of the signals results in the generation of a “measurement

table” that contains data on the ROI measurement (total and

average photon counts) and ROI information (dimension,

size, etc.).

4. Export the data and eventually proceed with statistical analysis

(see Note 13).

1. All experiments using mice must be performed according to

the applicable national guidelines and regulations. Diets of

laboratory rodents with low content of total protein, energy,

and/or p -aminobenzoic acid (PABA) can negatively in ﬂ uence

P. berghei infections (

14 ) . In our laboratory, we therefore pro-

vide diets with high protein content (20–25% of total and gross

energy content; 18,000–20,000 kJ/kg). If transgenic mice are

used, they should not express luciferase gene(s) that use

D -

luciferin as a substrate. Mice are kept under normal (day/

night) light conditions.

2. It is very important to dissolve inhibitors/antimalarial drugs

completely; vortexing and/or sonication and/or 37°C incu-

bation can help to dissolve compounds. The inhibitors in

stock solution can be diluted with DMSO or culture medium

according to their properties. It is better to make serial dilu-

tions fresh, though they can also be stored at −20 or −80°C.

We prepare stock solutions at a 100 times the ﬁ nal concentra-

tion required in the well so that when the drug is added to the

well the concentration of DMSO in overnight culture is <1%,

which is not harmful to the parasite development. No inhibi-

tion controls (i.e., culture without inhibitors) also contain

<1% DMSO.

3.2.4. Image Analysis

4. Notes

44130 Quantitative Analysis of Plasmodium berghei Liver Stages…

3. For luminescence measurements, we routinely use black-framed

microplates as they best reduce light scattering between wells,

which can arti ﬁ cially increase the signals detected in neighboring

wells and thereby calculated EC50 values.

4. Most manipulations with Huh7 cells and with genetically

modi ﬁ ed P. berghei parasites are performed in a Class II safety

cabinet.

5. It is important to maximize the release of sporozoites from the

dissected salivary glands. However, one should not homoge-

nize the salivary gland sample too much as this can reduce the

number of sporozoites. We therefore do the following: 10 s

vortexing, ten strokes of the homogenizer, 10 s vortexing.

6. Usually we check the plates regularly (once a day) under the

inverted microscope (10–20× magni ﬁ cation) for the cell

growth in these plates. Huh7 cells are usually collected for the

sporozoite assays when cells are 70–80% con ﬂ uent.

7. If you aim to analyze the effect of inhibitors on sporozoite

invasion, inhibitors can be added to the 24-well culture plates

during this step. If you aim at analyzing the effect speci ﬁ cally

on the development of the liver stages (after invasion of hepa-

tocytes by the sporozoites) inhibitors should be added during

Subheading

3.1.3 , step 5. See also Note 3 for more details on

adding inhibitors to the cultures.

8. Liver-stage development can be quanti ﬁ ed by measuring bio-

luminescence in lysed culture samples using a microplate reader

(see Subheading

3.1.4 , steps 1–7) or by measuring biolumi-

nescence directly in culture plates using the IVIS Lumina II

system. For direct imaging we directly add

D -Luciferin (80 mg/

ml) to the wells wait for 3 min and image the plates in the IVIS

Lumina II system in a comparable way as described for imag-

ing infected mice (see Subheading

3.2.3 , steps 7–9).

9. Usually mice are infected intravenously with de ﬁ ned numbers

(i.e., 10

to 10

) of freshly dissected sporozoites. However,

other studies may require sporozoites be administered by other

routes of administration. For intradermal inoculation we inject

50–100 m l (with varying numbers of parasites) of the sporozo-

ite suspension into the skin at various site of the body (most

often the neck). For subcutaneous inoculation we usually inject

500–800 m l (and varying numbers of parasites) of the sporozo-

ite suspension in the scruff of the neck.

10. Use the in vivo imaging system as recommended by the manu-

facturer. Ensure that the system is operational and that the

automatic background measurements have been performed

with settings that will be used for imaging the mice. Systems

are run by speci ﬁ c software that also serves as a guide to help

442 T. Annoura et al.

(inexperienced) users through the steps associated with

quantitative in vivo imaging and data analysis.

11. Reproducible imaging results are obtained with subcutaneous

injection of the substrate. However, we have evidence that

intravenous injection of

D - luciferin might improve the sensitiv-

ity of imaging, especially for organs that could degrade/elimi-

nate the substrate more rapidly or are less accessible for the

substrate.

12. Ensure that the ﬁ eld of view is set to provide an imaging area

that is wide enough to encompass the entire sample or the area

of interest. Distances of 10 cm are used for imaging a whole

body. Remove all dust particles from the sample stage. Mice

can be ﬁ xed by taping the legs to prevent them from moving

and interfering with the imaging of organs; black tape should

be used for ﬁ xing the mice to prevent background light

emission.

13. Bioluminescence imaging is simple to execute, allows monitor-

ing of the course of biological processes without killing the

experimental animal, and therefore reducing the number of

animals required for experimentation because multiple mea-

surements can be made in the same animal over time, minimiz-

ing the effects of biological variation. Usually, we use the mean

luminescent values of bodies/organs of three mice per imaging

time point.

Acknowledgment

TA, SC, and SMK received ﬁ nancial support of Top Institute

Pharma (The Netherlands) project: T4-102.

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