Lab tek 8 well chambered coverglass

Cells were seeded into a Lab-Tek™ 8-well Chambered Coverglass (Thermo Fisher Scientific). OGD was performed on the cells once they had reached confluency. Cells were fixed and permeabilized with ice-cold methanol and acetone for 10 min and blocked using 5% normal donkey serum (Sigma-Aldrich) in PBS for 1 h. The cells were incubated with primary antibodies (anti-ZO-1 and anti-claudin-5 antibodies) diluted in 1% normal donkey serum and then further incubated with secondary antibodies (Alexa Fluor 555-conjugated anti-rabbit) in 1% BSA. Fluorescence images were acquired and analyzed with a K1-Fluo confocal laser scanning microscope (Nanoscope Systems, Daejeon, Korea). To quantify the degree of TJs proteins, fluorescence images were analyzed with the software program, ImageJ.

COS-7, U2-OS, and HeLa cells (Cell Culture Facility, UC-Berkeley) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco 31053-028) supplemented with 10% fetal bovine serum (Corning), 1× GlutaMAX Supplement, and 1× non-essential amino acids, at 37 °C and 5% CO₂. For live-cell experiments, cells were cultured in Lab-Tek 8-well chambered coverglass (ThermoFisher), and transiently transfected with the above plasmids, either alone or in combination. Transfection was performed using Lipofectamine 3000 (ThermoFisher) or the Neon Transfection System (ThermoFisher), following the manufacturers’ instructions. For live-cell staining of the plasma membrane, lipid droplet, and DNA, wheat germ agglutinin (WGA) CF532 (300–500×, 29064, Biotium), LipidSpot488 (300–1000×, 70065, Biotium), and SYBR Green (15,000–100,000×, S7536, ThermoFisher) were added to the medium for 30 min at 37 °C and washed three times with DMEM before imaging. Imaging buffer was the regular culture medium with the addition of 25 mM HEPES at pH 7.4 (15630106, Gibco) or a commercial buffer based on MOPS (Hibernate A, BrainBits), with similar results observed.

Highly synchronous 3D7 parasite cultures at 4% hematocrit were diluted to 0.16% in warmed RPMI media and 0.2 mL of this was added to each well of a Lab-Tek 8-well Chambered Coverglass (Thermo Fisher Scientific). IgGs were added at concentrations indicated for each figure and the chamber was immediately placed on into a preheated (37°C) incubator stage of a Zeiss AxioObserver Z1 fluorescence microscope supplied with humidified gas (94% N₂, 1% O₂, and 5% CO₂). Late stage schizonts that appeared ready to rupture (Crick et al., 2013 (link)) were imaged with a Zeiss LCI Plan-NEOFLUAR 63x/1.3 DIC 1 mm Korr objective at 4 frames per second with an AxioCam MRm camera. The schizonts were imaged for 20 minutes and if they did not rupture, a new schizont in a new well was selected. The image files were cropped, time stamped and then converted to AVI video format in Zen microscopy software (Zeiss). The behavior of invading merozoites was manually viewed in FIJI and the invasion statistics were analyzed and graphed in Prism (Graphpad).

HEK cells
(ATCC) were grown in 25 mL cell culture flasks with filter caps (T-25,
Sarstedt) in Dulbecco’s modified Eagle medium (DMEM; Gibco)
supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin
(Gibco), 100 U/mL final concentration penicillin, and 100 μg/mL
streptomycin and maintained in a humidified atmosphere containing
5% CO₂ at 37 °C.
One day before transfection,
HEK cells were seeded in Lab-Tek 8-well chambered coverglass (Thermo
Fisher Scientific), with a seeding density of 1.0 × 10⁴ cells per well (volume of 400 μL). The cells were transfected
with 100 ng of peGFP-C1 or peGFP-GR-C1 plasmids for expression of
the enhanced green fluorescent protein (eGFP) or wild-type human GR
α fused with eGFP, respectively, using 0.2 μL of Lipofectamine
2000 (Thermo Fisher Scientific). Twenty-four hours after transfection,
the cell culture medium was replaced as described below, and the cells
were subjected to further analysis.
To induce GR translocation
into the nucleus, the synthetic ligand
dexamethasone (Dex; Sigma-Aldrich) was used. For pharmacological treatment,
a 2 mM Dex stock solution prepared by dissolving Dex in dimethyl sulfoxide
(DMSO) was diluted to 500 nM in phenol red-free medium FluoroBrite
DMEM (Gibco) and the cell culture medium was replaced. In control
experiments, the phenol red-free medium FluoroBrite DMEM (Gibco) was
used.