Cytomics fc500 mpl flow cytometer

GFP expression was monitored by flow cytometry of live cells. Bacteria were grown overnight at 37°C in LB with shaking, diluted into fresh medium (1:100), and incubated at 37°C to reach and O.D.₆₀₀ ∼ 2. For each sample, the GFP fluorescence of at least 50 000 cells was measured using a Cytomics FC500-MPl flow cytometer (Beckman Coulter, Brea, CA).

Apoptosis was measured by staining cells simultaneously with Annexin V–FITC (green fluorescence) and the non-vital dye propidium iodide (PI; red fluorescence), discriminating intact (Annexin V-FITC negative, PI negative), early apoptotic (Annexin V-FITC positive, PI negative), late apoptotic (Annexin V-FITC positive, PI positive) and necrotic cells (Annexin V-FITC negative, PI positive). The proportion of necrotic cells was always below 1%.
Flow cytometry analysis was carried out using the MitoStep + FITC Apoptosis Detection Kit, according to the manufacturer's instructions (Immunostep). Briefly, 7.5×10⁴ cells were seeded in Glu/Gal DMEM, apoptosis was induced by exposing them to 500nM Staurosporine for 24 hours and they were analyzed on a Cytomics FC500 MPL flow cytometer (Beckman Coulter). Assays were performed in duplicate in least three independent experiments.

Cells cultured in the presence of ACY-241 and/or paclitaxel were incubated for 60 min in the presence of 10 μM 5-ethynyl-2’-deoxyuridine (EdU) at 37°C. After EdU incorporation, cells were washed and resuspended in fixative solution using the Click-iT^® EdU Alexa Fluor^® 488 Flow Cytometry Assay Kit (Life Technologies, Grand Island, NY). Cellular DNA was stained using FxCycle™ Far Red Stain (Life Technologies) and cells analyzed on an Attune NxT Flow cytometer (ThermoFisher Scientific) or Cytomics FC 500 MPL flow cytometer (Beckman Coulter; Indianapolis, IN). Flow cytometry data were analyzed using FlowJo software (TreeStar; Ashland, OR). Dual-parameter plots were generated for Alexa Fluor^® 488–labeled EdU fluorescence (indicating newly synthesized DNA) and FxCycle™ Far Red Stain fluorescence (indicating relative DNA content). The generated plots have a typical inverted U-shaped pattern that identifies proliferating cells with bright EdU staining and nonproliferating cells with dim EdU staining that are either in G1 phase (with 2N DNA content) or in G2/M phase (with 4N DNA content). Cell death is indicated by the subG1 population, which are the nonproliferating cells with dim EdU staining and low DNA content (< 2N; corresponding to the lower left corner of the dual-parameter plot).

Cells were seeded into 6-well plates with 5 x10⁴ cells per well. After overnight incubation, cells were treated or not with EGFR inhibitors for 48 h. Adherent cells were dissociated by trypsin and collected by centrifugation at 500 g for 10 min. Cell pellets were washed twice with PBS, and cell membranes were disrupted by repeated cycles of freezing and thawing in liquid nitrogen. Then, cells were incubated with 200 μl of ribonuclease A (1 mg/ml) and stained with 200 μl of propidium iodide solution (100 μg/ml) (Sigma-Aldrich, St Louis, MO, USA). Fluorescence of cells was analyzed on a Cytomics FC 500 MPL Flow Cytometer (Beckman Coulter, Brea, CA, USA). Cell cycle distributions were calculated using ModFit LT 2.0 software (Verity Software House, Topsham, ME, USA).

The 3D7 P. falciparum clone and one Ghanaian patient isolate (BM057) were cultured in vitro (26 (link)) and were selected for DC4 PfEMP1 IE surface expression by repeated Ab selection as described previously (12 (link)). The identity of the isolates was routinely verified by genotyping as described previously (27 (link)), and Mycoplasma infection was regularly excluded using the MycoAlert Mycoplasma Detection Kit (Lonza) according to the manufacturer’s instructions.
P. falciparum IE were DNA-labeled with ethidium bromide and surface-labeled with mouse antisera obtained from the immunized mouse used for hybridoma production (15 μl serum/well), 24E9 mAb (100 μg/ml), or 24E9 Fab fragments (100 μg/ml). Whole Abs were labeled using an FITC-conjugated secondary anti-mouse IgG (1:100; Vector Labs), and an anti-mouse F(ab′)₂ IgG (1:100; Jackson Immunoresearch) was used to detect Fab fragments. FITC fluorescence data from ethidium bromide⁺ cells were collected on a Cytomics FC 500 MPL flow cytometer (Beckman Coulter) and analyzed in WinList version 6.0 (Verity Software House).

1 × 10⁶ cells were collected gently and incubated with trastuzumab (10 μg/ml) or control IgG at 4 °C for 30 min. After washing three times with PBS, the cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) at 4 °C for 30 min. After washing, cells were resuspended in PBS containing 1% formaldehyde. The fluorescence intensity was detected by Cytomics FC 500 MPL flow cytometer (Beckman Coulter, Fullerton, CA, USA) and analyzed using FlowJo software (Tree Star, Inc., CA, USA).