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Product Name:
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PfHDGFP
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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: PfHDGFP
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Material Provided:
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Each vial contains approximately 0.5 mL of P. falciparum-infected human blood in Glycerolyte 57 solution (1:5). Please refer to Appendix I for cryopreservation instructions.
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Packing/Storage:
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MRA-317 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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Safety Precautions:
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All blood cultures should be handled with appropriate safety precautions necessary for the handling of bloodborne pathogens. Personnel must be trained in accordance with their institutional policy regarding bloodborne pathogens
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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 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 RBCs to a 1% to 2% hematocrit solution (immediately or the next day).
9. Gently aerate culture with a 95% air, 5% CO2 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 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.
Please refer to Appendix I for cryopreservation instructions. |
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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. Kadekoppala, M., et al. “Stable Expression of a New Chimeric Fluorescent Reporter in the Human Malaria Parasite Plasmodium falciparum.” Infect. Immun. 68 (2000): 2328-2332. PubMed: 10722637.
2. Volkman, S. K., et al. “Excess Polymorphisms in Genes for Membrane Proteins in Plasmodium falciparum.” Science 4 (2002): 216-218. PubMed: 12364807.
3. Gardner, M. J., et al. “Genome Sequence of the Human Malaria Parasite Plasmodium falciparum.” Nature 419 (2002): 498-511. PubMed: 12368864.
4. Fried, M., et al. “Mass Spectrometric Analysis of Plasmodium falciparum Erythrocyte Membrane Protein-1 Variants Expressed by Placental Malaria Parasites.” Proteomics 4 (2004): 1086-1093. PubMed: 15048989.
5. Sanchez, B. A., et al. “Validation of a Plasmodium falciparum Parasite Transformed with Green Fluorescent Protein for Antimalarial Drug Screening.” J. Microbiol. Methods 69 (2007): 518-522. PubMed: 17466399.
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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 PfHDGFP, MRA-317, contributed by Kasturi Haldar.”
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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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