Zombie nir dye

Adherent cells at a density of 1×10⁶ cells per dish were stained for 30 min at 37°C, 95% humidity and 5% CO₂ either with 50 nM Mitotracker Green FM or 50 nM Mitotracker Orange CMXRos diluted in serum-free DMEM. Subsequently, cells were harvested and stained for 15 min at RT in the dark with 100 µl of ZOMBIE-NIR Dye (423105, BioLegend) diluted in 1× PBS 1:100. Cells were washed with cold 1× PBS with 2% FCS and analysed in 500 µl of 1× PBS with 2% FCS using FITC, PE and APC-A750 channels.

Immunofluorescence flow cytometry was performed as described previously^{3 (link)}. For mAb staining, the cells were washed with staining buffer (1% FBS in HBSS), resuspended in 50 μl of the same buffer, pre-incubated with purified anti-mouse CD16/32 (Biolegend) for 10 min, and incubated for 30 min at 4 ˚C with each fluorescence-conjugated mAb or isotype control matched with primary antibody. Zombie NIR™ dye (Biolegend) was used to assess live or dead status of cells. The samples were measured using a Gallios flow cytometry or CytoFLEX (Beckman Coulter). Doublets were distinguished from single cells by plotting FSC height vs FCS area. CD4⁺ T cells or Treg cells were sorted using a MoFlo XDP (Beckman Coulter). The purity of the sorted populations was more than 95%, as determined by a presorted sample run in parallel. Data were analyzed using in Kaluza analysis version 2.1 (Beckman Coulter).

Flow cytometry was used for analyzing the toxic effects of HCQ on lymphocytes with Zombie NIR™ Dye (Biolegend, San Diego, CA), and the frequencies of naïve CD4+T (CD4+CD62L+CD44-), Th1 (CD4+IFN-γ+), Th17 (CD4+IL-17A+), T regulator (Tregs) (CD4+CD25+Foxp3+) cells, and the expression of LOX-1 in RVECs. The cells were incubated with Fc block (clone 2.4G2, Bio Xcell) and stained with following antibodies from BioLegend: anti-mouse CD3 (BV421), anti-human CD3 (BV421), anti-human CD8 (PE), anti-human LOX-1 (APC), anti-mouse CD4 (Percp-cy5.5), anti-mouse CD25 (PE-cy7), anti-mouse CD44 (APC), anti-mouse CD62L (FITC), and anti-mouse CD69 (PE). For intracellular cytokine staining, the cells were stimulated with phorbol 12-myristate 13-acetate (PMA) (50 ng/mL; Sigma), ionomycin (500 ng/mL; Sigma), and Brefeldin A (1 µg/mL; Sigma) for 4–5 h. The The intracellular cytokines or transcription factor were stained with anti-human/mouse IFN-γ (BV786), anti-human/mouse IL-17A (BV650), and anti-human/mouse Foxp3 (FITC) after fixation and permeabilization. Data were analyzed using the FlowJo software 10.0 (Tree Star, Ashland, OR, USA).

Cells were incubated for 5 min at RT with Zombie NIR Dye (BioLegend, San Diego, CA, USA) to assess their viability. The Zombie NIR Dye was quenched, and cells were washed with cytometry buffer and blocked with FcR blocking reagent (1:50, Miltenyi Biotec, Bergisch Gladbach, Germany). Then, the samples were washed with cytometry buffer, stained with antibodies (Table 1) for 15 min at RT, and analyzed on a BD LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ) using the Software v10.6.1 (BD Biosciences, Franklin Lakes, NJ). Microglial cells were defined as CD45^lowCD11b⁺ and T cells as CD45^highCD11b⁻CD3⁺.

Naïve T cells (CD4⁺ CD25^- CD44^lo CD62L^hi) from spleen and peripheral lymph nodes were sorted on the MoFlow FACS sorter and stained with Cell Trace Violet (CTV) following manufacturer’s instructions. 5x10⁴ CTV⁺ cells per well were cultured in a 96-well plate with Dynabeads^® Mouse T-Activator CD3/CD28 (Invitrogen) at a 2:1 beads/cell ratio and complete RPMI medium for 4 days. Cells were recovered at 12, 24, 48, 72, and 96 hours and stained for the detection of CD4, CD25, CD44, and CD69 and viability. Zombie NIR dye (Biolegend) and antibodies were diluted in PBS and cells were incubated with the mix for 15 min at room temperature, washed, fixed, and acquired in the Attune NxT Cytometer (Thermo Fisher). Data analysis was done using FlowJo X software.
For the proliferation index, division index, and precursor frequency calculations, the number of cells of G0, G1, G2, G3 …. were obtained and the following algorithms were used:
Division index = # of divisions/# of cells at start of culture
Proliferation index = # of divisions/# of cells that went into division
Precursor frequency = (division index/proliferation index) * 100
where:
# of divisions = (G1/2)*1 + (G2/4)*2 + (G3/8)*3 ….
# of cells at the start of culture = G0 + (G1/2) + (G2/4) + (G3/8) ….
# of cells that went into division = (G1/2) + (G2/4) + (G3/8) ….

HSA-IL21 (the half-life extended IL21 was kindly provided by Anwita Biosciences, CA, USA), PD-1mAb (clone:J43), hamster IgG were purchased from Bioxcell company (catalog no. BE0091) for tumor therapy. For Flow cytometry, CD45 (clone: 30-F11), CD4 (clone: GK1.5), CD8 (clone: 53–6.7), NK1.1 (clone: PK136), B220 (clone: RA3-6B2), Foxp3 (MF-14), PD-1(29F.1A12), Tim-3 (clone: RMT3-23), Lag-3 (clone: C9B7W), CD39 (clone: 24DMS1), CD62L (clone: MEL-14), CD44 (clone: IM7), CD103 (clone: M290), CD69 (clone: H1.2F3), IFN-γ (clone: XMG1.2), GzmB (clone: QA16A02), CD11b (clone: M1/70), Ly6C (clone: HK1.4), Ly6G (clone: 1A8), Gr-1 (clone: RB6-8C5), CD24 (clone: M1/69), F4/80 (clone: BM8), CD11C (clone: N418), CD206 (clone: MR6F3), Arginase 1 (clone: A1exF5) were purchased from Biolegend ebioscience or BD Bioscience. Zombie NIR dye was purchased from Biolegend.

Single cells of SVF fraction were resuspended with PBS and stained with zombie NIR™ dye (#423105, Biolegend, USA), blocked with FcX Blocking CD16/32 (#156604), and dyed with a mix of surface marker antibodies. Cells were washed with cell staining buffer (#554657, BD Biosciences, USA), suspended, and transferred to a flow tube. Data was collected on a NovoExpress flow cytometer (Agilent, USA) and analyzed using Flowjo_V10 software (Tree Star).