Scribd
Upload
18 views

Materials and Methods

Uploaded by

Yuti Besain
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
18 views

Materials and Methods

Uploaded by

Yuti Besain
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
You are on page 1/ 6

Giemsa Staining and determination of Parasite

A blood smear was made, air dried and fixed with methanol. The slides were incubated for 15 to
20 minutes with Giemsa solution prepared by mixing 1 ml Giemsa stain and 4 ml Giemsa buffer
and washed with tap water. The slides were again air-dried and obsereved under a light
microscope.

The calculation of parasitemia, RBCs and parasites per field were counted and the parasitemia
was calculated using the following formula:

Percent Parasitemia = Total No. of parasites x 100

Average RBC’s x Total no. of fields

Transfer of Infection in Mosquitoes


Infected mice have optimal number of gametocytes were anesthesized and kept on the mosquito
cage for them to feed on the infected mice’s blood. The feeding procedure was performed for 20
minutes by changing the positions of the mice every 5 minutes. Prior to performing the feeding
procedure the mosquitoes are kept in a starving condition for 3-4 hours the feeding is then
performed twice, one in the evening after starvation and one in the next morning. Cotton pads
soaked in sucrose solution were used to maintain the nutrient conditions of the mosquitoes. The
infected mosquitoes are kept in environmental condition like 19ºC and 80% relative humidity.
The infection cycle was monitored further.

Isolation of salivary gland sporozoites


Mosquitoes were collected and kept in -20ºC for 2 minutes after which they become
unconscious. It is then first kept in ethanol only for a few seconds and it is removed right after.
Dulbecco’s Modified Eagle’s Medium (DMEM) was used to wash the mosquitoes. Three washes
were performed, 4 minutes each kept in ice. After the three washings the mosquitoes were
transferred in an eppendorf tube and the dissection of mosquitoes was then performed through
which the salivary glands were isolated.
Immunisation in C57BL/6 mice
The isolated salivary glands were collected in an eppendorf tube and DMEM was poured in it.
Using an ethanol wiped plunger the salivary glands were plunged thrice with first a 4 minute spin
and the rest two as short spins respectively after every 10 to 15 plunging. To count the number of
sporozoites a 50X dilution was made and 10µl of it was transferred to a haemocytometre to
count the number of sporozoites. All the quadrants are counted and an average is take out.

After counting the number of sporozoites/µl by the formula:

Number of sporozoites/µl = Average x Dilution x Haemocytometer Factor

Amount of sporozoites to be injected in the mice was calculated using the following formula:

Amount of sporozoites to be injected /mice = No. of sporozoites x No. of mice

Sporozoites/µl

Drug Cover for mice after Immunisation


The drug used for cover was Chloroquine (C18H26CIN3.2H3PO4 ). A diphosphate salt which is
already recognized to act upon blood stage parasites. It was prepared as per the number of mice
having each mouse to receive 0.8mg. The calculation was done accordingly.

In Vitro determination of Exo Erythrocytic Forms (EEF)


HepG2 cells were revived from the stock stored in liquid nitrogen, incubated at 37ºC and 5%
CO2. To determine EEFs the sporozoites were added to HepG2 cell culture to initiate the in vitro
culture. The cells were seeded a day before the infection. To prepare cells for infection the cells
were treated with trypsin-EDTA and then collected in complete DMEM medium. The cell
number is counted after making a dilution and using haemocytometer. Then they are seeded onto
the glass coverslips in a 48 well plate. Prior to seeding the cells the surface of the well plate were
treated with collagen; Type I solution from rat tail, diluted 1:9 times in MQ water. The salivary
gland sporozoites were added to HepG2 culture and the plate was spun at 310xg for 4 min and
incubated in a CO2 incubator. After 1 h, cells were washed with a medium containing
antibiotic/antimycotic and amphotericin B for 5 min. The medium was changed every 12 h and
the coverslips were taken out at different time points and fixed in 4% paraformaldehyde for 20
min at room temperature.

Immunoflorescence Assay
For localization experiments, transgenic parasites in the blood and liver stages were fixed at
various times using 4% PFA for 20 min at room temperature. 1% BSA-PBS was used to block
the cells after they had been permeableized by cooled methanol at 4°C for 15 minutes. EEFs
were stained with Upregulated in Infectious Sporozoite 4 (UIS4, 1:1000, Rabbit) and sera (1:50)
gathered from the immunised mice in order to see the parasite. For sporozoite stain TRAP 1:200)
and sera (1:50) from the immunized mice was used. Nuclei were stained with Hoechst 33342 and
the coverslips were mounted on Prolong diamond antifade reagent (Invitrogen). The images were
acquired in a using FV1000 software on a confocal laser scanning microscope (Olympus
BX61WI) using a UPlanSAPO 100x/1.4 immersion oil.

In vivo Imaging
We injected 3mg luciferene in each mice according to the body weight of 20g. With the help of
In Vivo Imaging System we took images with an exposure of 120 seconds each time.

Statistical analysis
The Luciferene assay was analysed by a spectral imaging software named AURA Imaging.

Retro-orbital Blood transfer


Blood smear of the mice was made, stained with Giemsa staining solution and then parasitemia
count of an average of 10 fields was performed. After the calculations the next step was to
collect blood from mice through its retro-orbital sinus using a heparinized Pasteur pipette and the
dilutions were made accordingly. After the dilutions were made the blood sample was injected
intravenously in three healthy mice, for each sample.
Perfusion and Isolation Immune Cells

Compositions:

1. Perfusion Buffer: 5-10 ml/ mouse


HBSS, 5mM HEPES, 0.5 mM EDTA
2. Wash Buffer: 50 ml/ mouse
PBS, 4% FBS, 0.5 mM EDTA
3. PBS Flow Buffer: 20 ml/mouse
1mM EDTA, 2% FBS
4. Collagen Solution: 5-10 ml/mouse
HBSS, 5mM HEPES, 0.5 mM CaCl2, 0.5 mg/ml collagenase.

Anesthetized the mouse by injecting the correct amount of anesthesia. Placed the mouse’s

belly on gauze pad and secured footpads in an X orientation. Disinfected the mouse using

70% ethanol and cut opened the skin to expose the peritoneal membrane. After exposing the

inner side of the mouse’s body we gently moved the intestines and stomach to the right side

and stuck the liver to the diaphragm. Identified the portal vein and the vena cava and using

sharp scissors we cut the portal vein. Collected the blood that was flowing near the portal vein

and transferred it to an eppendorf tube for sera collection. Turned on the pump and dripped the

perfusion buffer to the left side of the pump’s pipe. Catheterized the vein after which the liver

blanched. We cut the vena cava such as the blood and the buffer visibly flew through the vena

cava. The pump was stopped after the liver was completely blanched.

The pump was now stopped and the catheter was removed.

Then the line was shifted back for the perfusion of the next mouse.
Sample preparation for Fluorescence Acquired Cell Sorting

The liver was grabbed using forceps and the attachment between the liver and the diaphragm was
cut gently. The digested liver was transferred to the petri dish and 30ml cold wash buffer was
added to it. A rubber plunger was used to gently massage the liver rom over the strainer the
disperse the cells through it. The ell suspension was filtered in a 50ml conical tube. The cell
suspension was pelleted 600g for 10 minutes at 4ºC. It was then gently resuspended in 2.5 ml
PBS. After pelleting the cell suspension, the supernatant was discarded and the pellet was
strained with the help of using a strainer of pore size 70µm and PBS. The next step was to lyse
the RBC with the help of RBC lysis buffer for 2 minutes and centrifuged again. It was strained
again with the help of the same strainer and the volume make up was done by chilled PBS. The
filtrate was then spun at 600g for 10 minutes at 4ºC. The supernatant was discarded and the
pellet was dissolved in flow buffer.

An antibody cocktail was prepared using a multiple number of antibodies.

Catalogue Antibody Fluorochrome Stock Working


Number Concentration Concentration

2265468 CD3e FITC (0.5 mg/ml)


2262331 CD4 PE-Cy5 (0.2 mg/ml)
2331993 CD8a PE-Cy7 (0.2 mg/ml)
2197878 CD44 Alexa 700 (0.2 mg/ml)
2282646 CD62L APC (0.2 mg/ml)
2392637 CD183(CXR3-173) FITC (0.5 mg/ml)
2284176 KLRG1 Per-CP-eFlour 710 (0.2 mg/ml)
2452842
CD69 Per-CP Cy 5.5 (0.5 mg/ml)
The antibody cocktails made were as follows:

Cocktail 1: Lymphocytes

[CD3 + CD8 + CD4]

Cocktail 2: Effector Memory T cells

[CD3 + CD8 + CD44 + KLRG 1 + CD62L]

Cocktail 3: Regulator Memory T cells

[CD8 + CD44 + CD69 + CD183(CXR3-173)

The samples were made accordingly.

You might also like