Facs calibur 4 color flow cytometer

Change in the expression of α7nAChR on RAW264.7 cells was detected by flow cytometry (FACS). RAW264.7 cells were stimulated with 100 ng/mL LPS for 4 h, 8 h, and 20 h, respectively, and the cells incubated with PBS (volume equaled with the added LPS) for 4 h were used as the control. At the corresponding time points, the cells were rinsed with PBS, disassociated with nonenzyme cell detach solution, and transferred to tubes. After being centrifuged 5 min at 1500 rpm at 4°C and washed with PBS, the cells were fixed in 4% polyformaldehyde/PBS 30 min. Then the cells were washed with PBS for three times and suspended in 0.4 mL PBS. A mouse monoclonal antibody against α7nAChR (sc-374284, Santa Cruz Biotechnology Inc., USA) was added in the suspension (1 : 100), incubated overnight at 4°C, followed by washing with PBS, and stained with FITC-conjugated goat anti-mouse IgG (H + L) (EarthOx, USA) for 20 min at room temperature. The cells were washed with 2 mL PBS and resuspended in 0.4 mL PBS. All FACS data were analyzed on 10⁶ cells in FACSCalibur 4-color flow cytometer (BD Bioscience). The surface α7nAChR expression level was measured as the percent of positive cells.

Apoptosis was quantified by flow cytometry using the FITC-Annexin V/propidium iodide (PI) Apoptosis Detection Kit I (BD Pharmingen) according to the manufacturer's instructions. Data were collected using a FACS Calibur 4-color flow cytometer (BD Bioscience).

Enzymatic treatment of reticulocyte-enriched samples was performed by incubating 10 µl of the cell suspension with 1 mg/ml trypsin (from bovine pancreas, Sigma), 1 mg/ml chymotrypsin (from bovine pancreas, Sigma) and 0.5 U/ml neuraminidase (from Vibrio cholerae, Sigma) at 37 °C for 1 hour^{69 (link)}. CR1 enzymatic cleavage efficiency was tested by FACSCalibur 4-color flow cytometer (BD Biosciences)^{15 (link)} and neuraminidase treatment was assessed by performing an agglutination test using lectin from the peanut Arachis hypogaea, to detect T-antigen which is exposed after sialic acid cleavage^{69 (link)}.

For apoptosis assays, the FITC-Annexin V and propidium iodide (PI) Apoptosis Detection Kit I (BD Pharmingen) was used. For cell cycle analysis, PEL cell pellets were fixed in 70% ethanol, and incubated at 4°C overnight. Cell pellets were re-suspended in 0.5 mL of 0.05 mg/mL Propidium Iodide (PI) plus 0.2 mg/mL RNaseA and incubated at 37°C for 30 min. Cell cycle distribution was analyzed on a FACS Calibur 4-color flow cytometer (BD Bioscience).