Epics xl flow cytometer

Expression of the reporter gene eGFP in the tranfected cells was observed by fluorescence microscope (Nikon eclipse TE 2000), using the fluorescein isothiocyanate dichroic filter set as filter cube, (excitation at 450–490 nm; emission at 520 nm).
Approximately 3–5 × 10⁵ cells, after wash twice with 1x PBS, were analyzed on an EPICS-XL flow cytometer (Coulter, Miami, FL, USA). Approximately 5 × 10⁵ cells were washed and incubated with an anti-CD34-phycoerythrin (PE)-conjugated monoclonal antibody (Pharmingen, BD, Erembodegen, Belgium) for 30 min at 4 °C. Nonviable cells were excluded by gating based on propidium iodide (PI) (Pharmingen, BD). Transfected cells were sorted with a FACS Vantage (BD, Erembodegen, Belgium) as previously described^{8 (link),64 (link)}. Times and conditions for the estimation of eGFP and γ globin expression for each cell type is provided in the respective Results Section.

CRC cells with or without PFKP knockdown were collected and washed with PBS. Subsequently, the cells were stained with 50 mL of propidium iodide (Boehringer Mannheim Corp; Roche Molecular Biochemicals, Basel, Switzerland). Filtration was performed to remove cell aggregates, and the cell cycle was analyzed using a Coulter Epics XL Flow Cytometer.

The analysis of samples was carried out on an EPICS XL flow cytometer (Coulter, Hialeah, FL) equipped with an air-cooled argon laser tuned to 488 nm. The specific fluorescence signal corresponding to fluorescein isothiocyanate and H₂DCFDA was collected with a 525-nm band pass filter, and the signal corresponding to PI with a 620-nm band pass filter. A total of 10⁴ cells were scored in cell cycle assays, and 5×10³ cells in the H₂DCFDA assays.

eGFP expression of the transfected cells was examined and documented under a fluorescent microscope (Nikon Eclipse TE 2000). The filter cube used in the microscope was the fluorescein isothiocyanate dichroic filter set (excitation at 450–490 nm; emission at 520 nm). The software used for analysis was FlowJo 7.6 software (Tree Star, Ashland, OR).
Approximately 3–5 × 10⁵ cells were washed twice with 1x PBS and analyzed on an EPICS-XL flow cytometer (Coulter).
Approximately 5 × 10⁵ cells were washed and incubated with an anti-CD34-phycoerythrin (PE)-conjugated monoclonal antibody (Pharmingen) for 30 min at 4 °C. Dead cells were excluded by gating based on propidium iodide (PI) (Pharmingen). Transfected cells were sorted with a FACS Vantage (BD) as previously described19 (link).

Activated T-lymphocytes were induced to undergo apoptosis by UV-B irradiation for 30 s (90 J/cm²) in the presence or absence of 1 nM bortezomib (Santa Cruz, Heidelberg, Germany) or 10 µM Y27632 (Merck, Darmstadt, Germany). After 20 h, the amount of released LEVs was quantified by flow cytometry (FSC/SSC analysis), using an EPICS XL™ flow cytometer (Coulter, Hialeah, Fl, USA). Cell viability was assessed by AxV/PI staining as described above.

Detection of PDX cells in mouse samples was carried out with anti-human FITC-conjugated CD5 and PE-Cy5-conjugated CD7 antibodies (Coulter, Fullerton, CA, USA). Apoptosis was measured using Annexin V marker, as reported [23 (link)]. Surface Notch3 levels were analyzed using a PE anti-human Notch3 antibody (Biolegend, San Diego, CA, USA). Samples were analyzed on a Beckman Coulter EPICS-XL Flow Cytometer (Coulter) or a BD LSRII Flow Cytometer (BD Biosciences, San Jose, CA, USA).

Patients were assessed at weeks 4, 8, 12, 16, 20, and 24. Patients and those assessing clinical response (LSW and LW) remained blinded to the treatment. At each visit, history, physical examination and the ACR core set of disease-activity measures and DAS 28 scores were assessed. This included swollen joints count (66 joints), tender joint count (68 joints), patient’s assessment of pain on a scale from 0 (no pain) to100 (unbearable pain), patient’s global assessment of disease activity on a scale from 0 (disease inactive) to 100 (maximal disease activity), physician’s assessment of disease activity and patient’s assessment of physical function by health-assessment questionnaire (HAQ). Disease-activity score, DAS 28 (with physician’s assessment of 28 joints and patient’s self-assessment of disease activity), and the response according to the criteria of the European League Against Rheumatism (EULAR response) were also measured.
Laboratory monitoring included measurement of inflammatory markers, blood counts and routine biochemistry. Baseline and 24 week B cell counts, memory B cell (CD19 + 27+) percentages and T cell counts were measured using a Coulter Epics XL flow cytometer. Pneumococcal and tetanus antibody titers were measured at baseline and at 24 weeks by Enzyme linked immunosorbent assay (ELISA). Patients were followed for up to 48 weeks for adverse effects.