Facsc calibur

All participants provided blood specimens to measure CD4(+) T cell count at the starting point and during follow-up after switching treatment regimens, measured using flow cytometry (FACSC Calibur, BD). Real-time molecular beacon detection was applied to detect the viral load of HIV (NucliSens EasyQ Analyzer). Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify a 1300-bp fragment of the HIV pol gene for drug resistance mutation analysis and viral subtype determination. Successfully amplified sequences were analyzed for HIVDR using the Stanford University HIV Drug Resistance Database. All experimental protocols followed the manufacturer's instructions. People living with HIV with low or higher drug resistance to one or more drugs were considered as having HIVDR [26 (link)-28 (link)].

RSCL and rituximab resistant cell lines (RRCL) were treated with rituximab or Herceptin isotype control for 48h. Then the cells were stained with either FITC-conjugated mouse anti-human ICAM-1 (BD Invitrogen) or Isotype control (mouse IgG2α-FITC) (BD Invitrogen) for 30 minutes. Cells were then washed with PBS, fixed with 2% paraformaldehyde and analyzed by flow cytometry using FACSCCalibur (BD Biosciences). Cells were analyzed using the FCS express software (De Novo Software, Los Angeles, CA, USA).

Imaging. Mitochondria were immunolabeled with anti-SDHA antibody (ab14715, Abcam) and a conjugated Alexa Fluor-568 (2,124,366, Invitrogen); nuclei with DAPI. Cell imaging was performed with a confocal microscope (Leica DM5500 TCS SPE).
Flow cytometry. Cells were resuspended in PBS + 2 mM EDTA, labeled with 100 nM TMRM (T5428, Sigma-Aldrich) and analyzed with FACSCcalibur (BD Biosciences). Data were quantified with the FlowJo software (FlowJo, LLC).