Capacity of <i>P</i>. <i>berghei</i> GFP sporozoites per vial cryopreserved in CryoStor CS2 and post-thaw infection of HC-04 cells.

<p>Sporozoites from same lot mixed with CryoStor CS2 (1:3 ratio), aliquoted as 100 μl/vial with varying concentrations (0.25, 0.5, 1 million), cryopreserved, thawed, diluted to adjust same numbers and infected to HC-04 cells at 40,000 sporozoites/well. A sample of fresh sporozoites used to infect HC-04 prior to cryopreservation in medium alone served as control (solid line) to calculate the relative sporozoite infectivity, used to estimate viability.</p

Optimization of flow cytometer program.

<p>Hepatocytoma cells HC-04 seeded 80,000/well and infected with 40,000 sporozoites/well with fresh <i>P</i>. <i>berghei</i> GFP sporozoites or left uninfected. After 3 hrs HC-04 were stained with DRAQ5, harvested and analyzed by flow cytometer. (A) Uninfected and no stain HC-04 cells. (B) Uninfected and DRAQ5 stained cells. (C) Sporozoites infected and DRAQ5 stained cells. Stained cells were gated in FL-1A (GFP, 488 nm) and in FL-4A (DRAQ5, 640 nm).</p

Infectivity of fresh <i>P</i>. <i>berghei</i> GFP sporozoites to HC-04 cells.

<p>(A) The infectivity of HC-04 cells with fresh sporozoites is shown for increasing numbers of sporozoites, using 80,000 HC-04 cells per well. (B) The infectivity of HC-04 cells with fresh sporozoites is shown while varying the infection incubation time, using 40,000 sporozoites and 80,000 HC-04 cells per well.</p

Product information and important applications about cryogenic solutions.

<p>Product information and important applications about cryogenic solutions.</p

Infection, growth and development of <i>P</i>. <i>berghei</i> GFP sporozoites in HC-04 cells.

<p>Live images and IFA images of (A) fresh and (B) CryoStor CS2 cryopreserved <i>P</i>. <i>berghei</i> GFP sporozoites from day 1, day 2, day 3. Flow cytometer reading data from (C) fresh and (D) CryoStor CS2 cryopreserved <i>P</i>. <i>berghei</i> GFP sporozoites at 3 hrs, day 1, day 2, day 3. Stained cells were gated in FL-1A (GFP, 488 nm) and in FL-4A (DRAQ5, 640 nm).</p

Infectivity of fresh and CryoStor CS2 cryopreserved <i>P</i>. <i>berghei</i> GFP sporozoites in mice.

<p>Infectivity of fresh and CryoStor CS2 cryopreserved <i>P</i>. <i>berghei</i> GFP sporozoites in mice.</p

Summary of cryogenic solutions, post-thaw recovery and viability of <i>P</i>. <i>berghei</i> GFP sporozoites based on HC-04 cells infection.

<p>Summary of cryogenic solutions, post-thaw recovery and viability of <i>P</i>. <i>berghei</i> GFP sporozoites based on HC-04 cells infection.</p

Progression rate of fresh and CryoStor CS2 cryopreserved <i>P</i>. <i>berghei</i> GFP sporozoites invasion, growth and development in HC-04 cells.

<p>Progression rate of fresh and CryoStor CS2 cryopreserved <i>P</i>. <i>berghei</i> GFP sporozoites invasion, growth and development in HC-04 cells.</p

Maurer's clefts of are secretory organelles that concentrate virulence protein reporters for delivery to the host erythrocyte-1

<p><b>Copyright information:</b></p><p>Taken from "Maurer's clefts of are secretory organelles that concentrate virulence protein reporters for delivery to the host erythrocyte"</p><p></p><p>Blood 2009;111(4):2418-2426.</p><p>Published online 15 Feb 2009</p><p>PMCID:PMC2234068.</p><p>© 2008 by The American Society of Hematology</p>tanolysin (top) or saponin (bottom). Panels of fluorescent images show infected erythrocyte expressing HT-GFP, permeabilized with tetanolysin (top) or saponin (bottom), and probed with antibodies to GFP (green) and PfStomatin (red). Respective merged images are also shown. Dotted lines indicate erythrocyte periphery. Arrows show intraerythrocytic clefts. (B) 0° projections of an rHT-GFP–loaded erythrocyte ghost infected with 3D7 (top) or a mock-loaded erythrocyte ghost infected with transgenic parasite expressing HT-GFP (bottom). Empty arrowhead, cleft structure not labeled with intraerythrocytic rHT-GFP; solid arrowhead, GFP labeled cleft. (C) Cells in panel B fixed, permeabilized, and probed with antibodies to GFP (green) and resident cleft protein PfSBP1 (red). Arrows show clefts. (D) Immunoelectron microscopy of cells in panel B showing distribution of GFP associated with Maurer's clefts (MC). Bar indicates 500 nm. (E) Bar graph showing the percentage colocalization between GFP and Maurer's cleft in indicated samples by fluorescence microscopy. (F) Quantitation for the number of gold particles (measuring GFP) associated with clefts by immunoelectron microscopy over 20 infected erythrocytes. In all fluorescence micrographs: p, parasite (nucleus stained with Hoechst 33342; blue); ec, erythrocyte cytosol; bar, 2 μm

Maurer's clefts of are secretory organelles that concentrate virulence protein reporters for delivery to the host erythrocyte-6

<p><b>Copyright information:</b></p><p>Taken from "Maurer's clefts of are secretory organelles that concentrate virulence protein reporters for delivery to the host erythrocyte"</p><p></p><p>Blood 2009;111(4):2418-2426.</p><p>Published online 15 Feb 2009</p><p>PMCID:PMC2234068.</p><p>© 2008 by The American Society of Hematology</p>throcytic structure, possibly a cleft. (B) Immunoelectron micrographs of trophozoite parasite (p)-infected cells expressing HT-GFP. Ultrathin sections were probed with antibodies to GFP and secondary antibody gold (10 nm) conjugate. Arrows indicate gold particles at intraerythrocytic Maurer's clefts (MC). No gold labeling was detected in absence of primary antibody or when a nonspecific primary was used (not shown). Bar, 200 nm. (C) Single optical section of an infected erythrocyte expressing HT-GFP. Samples were treated to release soluble GFP, fixed, and probed with antibodies to GFP (green) and Skeletal Binding Protein1 (PfSBP1, red). Arrow, GFP labeled cleft structures at the periphery of infected erythrocyte; arrowheads, clefts proximal to the parasite. In fluorescence micrographs, p denotes parasite nucleus stained with Hoechst 33342; bar, 2 μm. Schematic representation of the construct is indicated above with ER-type signal sequence (red), sequence containing HT signal (blue) fused to GFP (green) and myc (orange)