Lsr fortessa flow cytometer with high throughput sampler

For 40 h.p.i. time-point, tightly synchronous parasites were enriched using 63% Percol, washed twice with incomplete RPMI medium, and processed immediately on the 10X Chromium controller (10X Genomics, Pleasanton, CA). For 16 h.p.i. time point parasites were stained with Mitotracker Deep Red FM (Life Technologies, #M22426) for FACS analysis and flow sorting, respectively. Briefly, 50 μl of SYBR Green I stained RBCs were analyzed on BD LSR Fortessa Flow Cytometer with High Throughput sampler (BD Biosciences, San Jose, CA, USA) using BD FACS Diva Software v6.2 and 488 laser excitation / 530 emission filter to determine the concentration of SYBR Green I positive cells per microliter. A BD Influx Cell Sorter (BD Biosciences, San Jose, CA, USA) with BD FACS Sortware v1.0.01 software was used to sort ~40,000 Mitotracker Deep Red FM - positive RBC’s using a 70 μm nozzle, a 640 nm laser excitation / 670 nm emission filter and a pressure setting of 30 psi. Post-sorted cell concentration and quality were checked using a Countess^® II Automated Cell Counter (Invitrogen) and FLoid^™ Cell Imaging Station (ThermoFisher). Finally, labeled cells (i.e., SYBR Green I or Mitotracker Deep Red FM positive cells) were then loaded onto a 10X chip (Chip G) and processed immediately on the 10X Chromium controller (10X Genomics, Pleasanton, CA).

For the 40 h.p.i. timepoint, tightly synchronous parasites were enriched using 63% Percoll, washed twice with incomplete Roswell Park Memorial Institute 1640 medium, and processed immediately on the 10X Chromium controller (10X Genomics). For the 16 h.p.i. timepoint, parasites were stained with Mitotracker Deep Red FM (Life Technologies, #M22426) for FACS analysis and flow sorting, respectively. Briefly, 50 µl of SYBR Green I stained RBCs were analysed on BD LSR Fortessa Flow Cytometer with High Throughput sampler (BD Biosciences) using BD FACS Diva Software v6.2 and 488 laser excitation/530 emission filter to determine the concentration of SYBR Green I positive cells µl⁻¹. A BD Influx Cell Sorter (BD Biosciences) with BD FACS Sortware v1.0.01 software was used to sort ~40,000 Mitotracker Deep Red FM-positive RBCs using a 70 µm nozzle, a 640 nm laser excitation/670 nm emission filter, and a pressure setting of 30 psi. Post-sorted cell concentration and quality were checked using a Countess II Automated Cell Counter (Invitrogen) and FLoid Cell Imaging Station (Thermo Fisher Scientific). Finally, labelled cells (that is, SYBR Green I or Mitotracker Deep Red FM-positive cells) were then loaded onto a 10X chip (Chip G) and processed immediately on the 10X Chromium controller (10X Genomics).