4. Analysis of Apoptosis in RAS and p36p
À Parasite-Invaded
Hepatocytes (In vitro): Detection of apoptosis is done by active
caspase-3 detection [52] and nucleus is stained by DAPI as
described [52]. 3 Â 10
4 sporozoites of WT, p36p
À , or RAS,
gamma source, 16 Krad [24], are added to 2 Â 10
5 HepG2
cells [23, 24] and incubated for 6 h before staining with antiHSP70 Ab [23] for detection of parasites. Apoptotic cells are
detected by using an active caspase-3 detection kit (Promega)
and DAPI staining after 6 h of infection. Infectious and apoptotic cells are evaluated by counting the number of parasiteinfected cells and apoptotic parasite-infected cells per coverslip
in triplicate, respectively.
5. Analysis of Apoptosis in p36p
À Parasite-Invaded Hepatocytes
(In Vivo): Intravenous injection containing 5 Â 10
5 p36p
À or
WT sporozoites is administered to two groups of C57BL/6
mice. Livers are extracted 6 h postinfection. Parasites are
detected using anti-HSP70 [23] in the tissue histological sections of each mouse. Nuclear morphology is visualized by
DAPI staining. Sections are examined for the presence of
infected cells with apoptotic signs.
6. Analysis of the Immunization Potential of p36p
À Sporozoites:
BALB/c and C57BL6 mice are subjected to intravenous injection of p36p
À sporozoites or RAS [24] or PBS. Parasitemia is
detected in Giemsa-stained blood smears. Mice are evaluated
for blood stage parasitemia in alternate day from day 3 to day
21 after challenge with varying dosage of WT sporozoites at
different time points. Euthanization is performed 40 h after
parasitic challenge. Liver is extracted and quantified for infection by real-time PCR quantification [39].
3.8 Gamma
Irradiation
for Mutagenesis
in Parasites [25]
1. Collection of Sporozoites: Sporozoites are collected by dissecting the salivary glands of laboratory bred Anopheles stephensi.
Prior to dissection, A. stephensi are fed on a hamster infected
with Plasmodium berghei for 14 days.
2. Homogenization of salivary glands: Isolated salivary gland is
homogenized in 50% human plasma diluted in saline.
3. X-ray irradiation: The homogenized sporozoites are subjected
to irradiation of 280 kVp X-ray beam of a Picker Vanguard
teletherapy unit. The radiation range varied from 2 to 15 krads.
4. Protective Immunity Assessment: A/J mice are injected intraperitoneally with approximately 5000–7000 sporozoites
contained within 0.2 ml injection volume. Same-sex and
age-matched A/J control mice are subjected to injection containing same volume nonirradiated sporozoites. Vaccinated and
control mice are subjected with secondary challenge of viable
sporozoites (1000/mouse) about 2 weeks after injection of the
irradiated sporozoites.
352
Dipasree Hajra et al.
À Parasite-Invaded
Hepatocytes (In vitro): Detection of apoptosis is done by active
caspase-3 detection [52] and nucleus is stained by DAPI as
described [52]. 3 Â 10
4 sporozoites of WT, p36p
À , or RAS,
gamma source, 16 Krad [24], are added to 2 Â 10
5 HepG2
cells [23, 24] and incubated for 6 h before staining with antiHSP70 Ab [23] for detection of parasites. Apoptotic cells are
detected by using an active caspase-3 detection kit (Promega)
and DAPI staining after 6 h of infection. Infectious and apoptotic cells are evaluated by counting the number of parasiteinfected cells and apoptotic parasite-infected cells per coverslip
in triplicate, respectively.
5. Analysis of Apoptosis in p36p
À Parasite-Invaded Hepatocytes
(In Vivo): Intravenous injection containing 5 Â 10
5 p36p
À or
WT sporozoites is administered to two groups of C57BL/6
mice. Livers are extracted 6 h postinfection. Parasites are
detected using anti-HSP70 [23] in the tissue histological sections of each mouse. Nuclear morphology is visualized by
DAPI staining. Sections are examined for the presence of
infected cells with apoptotic signs.
6. Analysis of the Immunization Potential of p36p
À Sporozoites:
BALB/c and C57BL6 mice are subjected to intravenous injection of p36p
À sporozoites or RAS [24] or PBS. Parasitemia is
detected in Giemsa-stained blood smears. Mice are evaluated
for blood stage parasitemia in alternate day from day 3 to day
21 after challenge with varying dosage of WT sporozoites at
different time points. Euthanization is performed 40 h after
parasitic challenge. Liver is extracted and quantified for infection by real-time PCR quantification [39].
3.8 Gamma
Irradiation
for Mutagenesis
in Parasites [25]
1. Collection of Sporozoites: Sporozoites are collected by dissecting the salivary glands of laboratory bred Anopheles stephensi.
Prior to dissection, A. stephensi are fed on a hamster infected
with Plasmodium berghei for 14 days.
2. Homogenization of salivary glands: Isolated salivary gland is
homogenized in 50% human plasma diluted in saline.
3. X-ray irradiation: The homogenized sporozoites are subjected
to irradiation of 280 kVp X-ray beam of a Picker Vanguard
teletherapy unit. The radiation range varied from 2 to 15 krads.
4. Protective Immunity Assessment: A/J mice are injected intraperitoneally with approximately 5000–7000 sporozoites
contained within 0.2 ml injection volume. Same-sex and
age-matched A/J control mice are subjected to injection containing same volume nonirradiated sporozoites. Vaccinated and
control mice are subjected with secondary challenge of viable
sporozoites (1000/mouse) about 2 weeks after injection of the
irradiated sporozoites.
352
Dipasree Hajra et al.