Rpmi media

Jurkat E6.1 cells (obtained from ATCC) were cultured in RPMI media (GIBCO) containing 10% heat inactivated fetal calf serum to a maximum density of 2 × 10⁶ cells/ml.

For BM chimeras, acidic drinking water (pH 2,5–3,0) was given for 8 days to B6 x B6.SJL F1 (CD45.2⁺ CD45.1⁺) recipient mice prior to irradiation with a lethal single dose of 800 rad (TH780C - Theratronics, Canada).The day after irradiation, BM cells were transferred and mice were treated with neomycin sulfate (2 mg/mL) in the drinking water for 15 days. Marrow was harvested from the femur and tibia of B6.SJL (CD45.1⁺) and B6, Myd88^−/−, Il1r1^−/−or Il18r1^−/− (CD45.2⁺) mice by flushing with cold RPMI media (GIBCO). BM cells were washed thoroughly with PBS and 4 × 10⁶ total cells were injected into the irradiated recipient mice. For mixed chimeras, CD45.1⁺ and CD45.2⁺ BM cells were transferred in a 1:1 ratio (2 × 10⁶ cells each). Alternatively, non-irradiated mixed chimeric Rag2^−/− mice were generated by the iv injection of 20 × 10⁶ spleen cells from WT B6.SJL (CD45.1⁺) and Myd88^−/− (CD45.2⁺) mice in a 1:1 ratio. Six to eight weeks after the first reconstitution, Rag2^−/− mice received a second injection of WT B6 x B6.SJL F1 (CD45.1⁺CD45.2⁺) and Myd88^−/− (CD45.2⁺) splenocytes in a 1:1 ratio, 6 hr before being infected. In all experiments, mice were infected 6 to 8 weeks after cell transfer, when reconstitution was achieved.

B6 ALL cells were a kind gift of Dr Nidal Boulos (St. Jude Children's Research Hospital, Memphis, TN [11 (link)]). All human cell lines were maintained in RPMI media (Gibco, Carlsbad, CA) supplemented with 10% FBS, penicillin and streptomycin. Cells were grown at 37°C with 5% CO₂. All murine cells were maintained in B cell media (45% DMEM, 45% IMDM, 10% FBS, supplemented with penicillin and streptomycin). Viability assays were done using the Cell Titer-Glo assay (Promega, Madison, WI) according to the manufacturer's protocol or by Trypan blue exclusion assay using the Countess cell counting system (Invitrogen, Carlsbad, CA). All viability assays were done in triplicate in at least three separate experiments (at least 9 values for each concentration tested).

Entire open reading frame of PvTRAg36.6 (960 bp) and PvTRAg56.2 (1394 bp) genes were custom synthesized commercially (Biolinkk, New Delhi, India) and were cloned into transfection vector pARL1a-GFP which expresses genes under the control of Pfcrt (P.falciparum chloroquine resistant transporter) promoter, in KpnI and BglII cloning sites to generate transgenic constructs: pARL1a-PvTRAg36.6 and pARL1a PvTRAg56.2. The PvTRAg56.2 gene was also cloned in pARL1a-GFP vector consisting of late stage promoter, pfama1 (P.falciparum apical membrane antigen).
Plasmodium falciparum 3D7 parasites were cultured with human erythrocytes at 4% haematocrit in RPMI media (Gibco Co, Auckland, Grand Island, USA) supplemented with 5% Albumax I (Gibco) following Trager and Jensen (Trager and Jensen, 1976). Synchronized ring stage parasites were transfected with 100 μg of each recombinant plasmid [33 (link)] by electroporation (310V, 950 μF). Parasites were maintained under the 5 nM WR99210 drug pressure. Resistant parasites appeared after one to two months of transfection.
To ascertain the expression of PvTRAg-GFP fusion proteins in the transgenic line, parasite lysates were subjected to 10% SDS-PAGE and western blot analyses using monoclonal anti-GFP antibody (1:2000 dilution).