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Product Name:
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Plasmodium falciparum, Strain PM2GT
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Manufacturer:
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BEI Resources
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Taxonomy:
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Protozoa Classification: PIasmodiidae, Plasmodium Species: Plasmodium falciparum Strain: PM2GT (also referred to as clone B7)
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Additional Information:
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MRA-805 could provide a better understanding of plasmepsin trafficking and maturation. PM II is an aspartic protease, contributing to the degradation of hemoglobin, which is essential for the growth of the intraerythrocytic stages of P. falciparum. It is localized in the food vacuole, cytostome and endoplasmic reticulum and is considered a promising target for antimalarial drug development.3,5
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Material Provided:
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Each vial of MRA-805 contains approximately 0.5 mL of P. falciparum-infected human blood in Glycerolyte 57 solution (1:5). Please see Appendix I for cryopreservation instructions.
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Packing/Storage:
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MRA-805 was packaged aseptically in cryovials. The product is provided frozen and should be stored at -80°C or colder immediately upon arrival. For long-term storage, the vapor phase of a liquid nitrogen freezer is recommended (-130°C or colder). Freeze-thaw cycles should be avoided.
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Growth Conditions:
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RPMI 1640 medium adjusted to contain 10% (v/v) heat-inactivated human serum (pooled Type A), 25 mM HEPES, 2 mM L-glutamine, 2 g/L D-glucose, 27 µg/mL hypoxanthine and 5 µg/mL gentamicin (optional)
Human serum (pooled Type A or Type O recommended)
Please see Appendix II for complete medium preparation instructions and notes.
Incubation:
Temperature: 37°C
Atmosphere: 90% N2,
5% CO2, 5% O2
Propagation:
1.
Place the frozen vial in a 37°C water bath until the culture is completely
thawed. Transfer the vial to a
biological safety hood and wipe the outside surface of the vial with 70%
ethanol.
2.
Using a sterile 1 mL pipette, aseptically transfer
the contents of the vial to a sterile 50 mL conical centrifuge tube.
3.
Add 12% sodium chloride (NaCl) solution dropwise,
approximately 1:5 ratio NaCl to cell mixture (0.2× original culture
volume). Allow it to stand for 5
minutes.
4.
Using a 1 mL syringe and 27-gauge needle, add
dropwise while shaking 10 volumes of a 1.6% NaCl solution (10:1 ratio NaCl to
original culture volume).
5.
Centrifuge at 1000 × g for 5 minutes and remove
most of the supernatant, leaving approximately 0.5 mL to 1 mL to resuspend the
cell pellet. Resuspend the cells by
gently swirling the tube.
6.
Add dropwise while shaking 10 volumes of complete
medium. Centrifuge at 1000 × g for 5
minutes and carefully remove the supernatant.
7.
Add 5 mL of complete medium and transfer the sample
to a 25 cm² tissue culture flask.
8.
For continuous culture, add uninfected red blood
cells (RBCs) to a 1% to 2% hematocrit solution (immediately or the next day).
9.
Gently aerate culture with a 90% N2, 5%
CO2, and 5% O2 gas mixture through a sterile, cotton-plugged Pasteur
pipette. Incubate the flask at 37°C.
10. Take a smear for
Giemsa staining after 1 day to evaluate parasite growth and determine
parasitemia.
Maintenance:
Note: Changing of the culture medium every 1 day is
required for malaria-infected erythrocyte cultures.
1.
Remove the flask with infected culture from the
37°C incubator and place it onto a flask warmer.
2.
Carefully remove the supernatant with a sterile,
unplugged Pasteur pipette under vacuum.
Remove as much of the supernatant as possible without taking the cells.
3.
Add 25 mL of sterile warm (37°C) complete medium to
the flask, gently mix and aerate, then quickly tighten the cap and place the
flask in the 37°C incubator until the next change of medium.
Preparation of Blood Smear:
1.
Carefully remove 0.5 mL to 1 mL of mixed culture
with a sterile pipette and transfer to a microcentrifuge tube.
2.
Centrifuge the microcentrifuge tube at high speed
and aspirate the supernatant.
3.
Mix the pellet and transfer 6 µL of the suspension
to a glass slide for a thick film smear or 2 µL for a thin film smear. Spread the drop into a thin film using the
edge of a clean glass slide. Air dry for
3 minutes at room temperature.
4.
Fix the blood smear by rinsing it with methyl
alcohol. Air dry for 3 minutes at room
temperature.
5.
Stain blood films in 10% Giemsa solution for 15
minutes. Rinse with distilled water and
allow to air dry.
6.
Using light microscopy at 100× magnification,
determine parasitemia of culture.
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Disclaimers:
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You are authorized to use this product for research use only. It is not intended for human use. Use of this product is subject to the terms and conditions of the BEI Resources Material Transfer Agreement (MTA). The MTA is available on our Web site at www.beiresources.org. While BEI Resources uses reasonable efforts to include accurate and up-to-date information on this product sheet, neither ATCC® nor the U.S. Government makes any warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. Neither ATCC® nor the U.S. Government warrants that such information has been confirmed to be accurate. This product is sent with the condition that you are responsible for its safe storage, handling, use and disposal. ATCC® and the U.S. Government are not liable for any damages or injuries arising from receipt and/ or use of this product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, the U.S. Government, ATCC®, their suppliers and contributors to BEI Resources are not liable for damages arising from the misidentification or misrepresentation of products. |
References:
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1. Goldberg, D. E.,
Personal Communication.
2. Klemba, M., et al.
“Trafficking of Plasmepsin II to the Food Vacuole of
the Malaria Parasite Plasmodium falciparum.” J. Cell Biol.
164 (2004): 47-56. PubMed: 14709539.
3. Banerjee, R., et al. “Four Plasmepsins are Active in the Plasmodium falciparum Food Vacuole, Including a Protease with an Active-Site
Histidine.” Proc. Natl. Acad. Sci. USA 99 (2002): 990-995. PubMed:
11782538.
4.
Walliker, D., et al. “Genetic Analysis of the Human
Malaria Parasite Plasmodium falciparum.”
Science 236 (1987): 1661-1666.
PubMed: 3299700.
5.
Moura, P.A., J.B. Dame and D.A. Fidock. Role of Plasmodium
falciparum Digestive Vacuole Plasmepsins in the Specificity and Antimalarial
Mode of Action of Cysteine and Aspartic Protease Inhibitors. Antimicrob.
Agents. Chemother. 53 (2009) :4968-4978. PubMed: 19752273.
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Citation:
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Acknowledgment for publications should read "The following reagent was obtained through BEI Resources, NIAID, NIH: Plasmodium falciparum, Strain PM2GT, MRA-805, contributed by D. E. Goldberg."
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Biosafety Level:
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2
Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health. Biosafety in Microbiological and Biomedical Laboratories (BMBL). Current Edition. Washington, DC: U.S. Government Printing Office.
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