Lsr 2 hts flow cytometer

Mouse CD4⁺ T cells were isolated from spleens of WT and Ctr1^+/- mice using the mouse CD4⁺ T-cell isolation kit from Miltenyi Biotec (Bergisch Gladbach, Germany) according to the manufacturer’s protocol. We routinely obtained > 95% CD4⁺ T cells as assessed by FACS. Whole-cell patch clamping was then performed following stimulation with anti-CD3 (5 µg/ml) and anti-CD28 (2 µg/ml) antibodies for 48 hr, as described (Srivastava et al., 2008 (link)). For intracellular cytokine staining, cells were rested overnight and then re-stimulated with anti-CD3 and -CD28 antibodies for 4 hr in the presence of brefeldin-A (1 µg/ml, Sigma-Aldrich). Intracellular staining was performed using the Fixation/Permeabilization kit (BD Biosciences, Franklin Lakes, NJ, Cat # 554714) and antibodies to: IL-2 (Cat # JES6-5H4), IFN-γ (Cat # XMG1.2) or TNFα (Cat # MP6-XT22), according to the manufacturer’s (eBioscience, San Diego, CA) instructions. Cells were analyzed on a LSRII HTS flow cytometer (BD Biosciences), and the results were analyzed with Treestar (FlowJo, Ashland, OR).

72 h and 120 h post-transfection medium was collected and adherent U87R cells in monolayer culture harvested, washed twice with sample buffer (100 mg glucose; 100 mL PBS without Ca²⁺ or Mg²⁺) and fixed in 70% (v/v) cold ethanol. Cells were pelleted, washed once with sample buffer and resuspended in propidium iodide (PI) solution (50 μg mL⁻¹ PI, 0.5 mg mL⁻¹ RNase in sample buffer, pH 7.4) for 30 min in the dark. The data from 10,000 cells per sample were collected and analysed using the BD FACSDiva program on a LSR II HTS flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA).

KCa3.1 containing a hemagglutinin (HA) epitope tag inserted into the extracellular loops between transmembrane helices S3 and S4 was transfected into Ctr1^-/- and WT MEFs. To assess the plasma membrane levels of KCa3.1, cells were fixed with 4% paraformaldehyde for 10 min on ice and washed three times with PBS. The non-permeabilized fixed cells were incubated with anti-HA antibody (1:200 dilution) for 1 hr on ice, washed three times with PBS, and stained with secondary anti-mouse FITC-conjugated antibody (1:500 dilution) for 30 min on ice. Cells were then washed three times with PBS prior to analysis by flow cytometry. Cells incubated with only the secondary antibody served as the negative control. Flow cytometry was performed using LSR II HTS flow cytometer (BD Biosciences), and the results were analyzed using Treestar (FlowJo).