Facsaria sorp cell sorter

PBMCs of B‐CLL patients and healthy donors were derived from whole blood by Ficoll gradient centrifugation. Single cell suspensions were prepared from fresh human tonsil specimens using a MediMachine (BD, USA) and filtered through a 40‐μm cell filter. Surface staining was performed with the monoclonal antibodies anti‐CD5 FITC (clone DK23, DAKO, USA), anti‐CD19 PE‐Cy5 (clone HIB19, eBioscience, USA), anti‐CD23 PE (clone MHM6, DAKO, USA), FMC7 FITC (clone MCA792F, Serotec, USA), anti‐CD20 PE‐Cy5 (clone 2H7, eBioscience, USA), and anti‐κ light Ig chain and anti‐λ light Ig chain (κ‐FITC/λ‐PE, AHM4907, Invitrogen, USA). The cells were incubated with the antibodies at +4°C for 20 min, washed with PBS, and analyzed on a FACSAria SORP cell sorter (BD Biosciences, USA). Diva software (BD Biosciences, USA) was employed for the data analysis.
All samples were kept at +4°C during staining and prior to FACS analysis. Primary FACS profiles were obtained for each specimen on the day of retrieval typically in 4 h after extraction. Aliquots of cells were frozen in FBS with 10% DMSO in liquid nitrogen for further FACS sorting.

MSC and EV fractions were characterized using a FACS Aria SORP cell sorter equipped with 405 and 355, 488, 561, and 640 nm lasers (BD Biosciences, San José, CA, USA). Staining of MSCs was performed with fluorescein isothiocyanate (FITC)-conjugated annexin V (Sigma-Aldrich, St. Louis, MO, USA) as a marker for phosphatidylserine. Staining of MSC-derived EVs was performed with FITC-annexin V in combination with a PKH26 dye (Sigma-Aldrich, St. Louis, MO, USA) as a marker for lipid membranes. Non-stained MSCs/EVs and single-stained EVs with PHK26 were used as controls. Specimens were sampled for 3 min at a flow rate of 30 µL per min and data were analyzed using Flowing Software (Turku Centre for Biotechnology, Turku, Finland). Since phosphatidylserine is believed to be a marker of apoptotic cells, we analyzed MSCs after serum deprivation for 24 h as a positive control for apoptosis.

Human or mouse liver single-cell suspensions were incubated with anti-DLK-FITC (MBL, D187-4, 1:100), anti-EpCAM-APC (eBioscience, 17-5791-82, 1:50), anti-NCAM1 (Sangon Biotech, D198946, 1:50), Alexa Fluor 647 donkey anti-mouse IgG (Invitrogen, A31571, 1:1000), anti-cKit-APC (BioLegend, 105812, 1:200), or anti-CD71-PE (eBioscience, 113808, 1:200) antibodies at 4 °C for 20 min. After washing once with PBS, cells were analyzed and sorted using a FACS Aria SORP cell sorter (BD Biosciences). Dead cells were excluded by DAPI staining.

Cell death was analyzed by flow cytometry using a FACSAria SORP instrument (BD Biosciences, San Jose, CA, USA). Cells were simultaneously stained with 1 μg/ml Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA), 100 nM TMRE (tetramethylrhodamine, ethyl ester, perchlorate, Thermo Fisher Scientific, Waltham, MA, USA) and annexin V-Alexa Fluor 647 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Thus, the DNA content, mitochondrial membrane potential and phosphatidylserine externalization were analyzed together for each sample. The detection parameters were as follows: Ex. 405 nm, Em. 450/50 BP for Hoechst 33342; Ex. 561 nm, Em. 585/15 BP for TMRE and Ex. 647 nm, Em. 640/14 BP for annexin V-Alexa Fluor 647. Additionally, the cells were stained with anti-caspase 3 PE-conjugated antibodies (PE active caspase-3 apoptosis kit; BD Biosciences, San Jose, CA, USA). Cell fixation, permeabilization and staining were performed according to the manufacturer’s instructions. Active caspase 3-PE was detected at Ex. 561 nm, Em. 585/15 BP using a FACSAria SORP cell sorter (BD Biosciences, San Jose, CA, USA).

Lentivirus was produced by co-transfection of 293T cells with 3 μg luciferase expressing vector pLV-mCherry-P2A-luciferase, 2 μg Δ8.9 HIV-1 packaging vector, 1 μg VSVG envelope glycoprotein vector, and 2.5 μl/μg Lipofectamine 2000 (Invitrogen) in Opti-MEM media (Gibco) in 6-well plates at 37 °C 5% CO₂ for 16 h. Supernatant virus was collected, 0.45 μm filtered, and diluted 1:5 and supplemented with 8 μg/ml polybrene (Santa Cruz Biotechnology) before spinfecting Myc-CaP cells for 2 h at 32 °C. At least 48 h later, mCherry positivity was verified and sorted for top 10% positivity using a FacsAria SORP cell sorter (BD).

To generate a genetic knockout of PHAROH, two sgRNAs targeting the promoter region were combined, creating a deletion including the TSS. Guide design was performed on Benchling (https://benchling.com) taking into account both off-target scores and on-target scores. The sgRNA targeting the gene body of PHAROH was cloned into a pSpCas9(BB)−2A-GFP vector (PX458, Addgene plasmid #48138), and the sgRNA targeting the upstream promoter region was cloned into a pSpCas9(BB)- 2A-mCherry vector. Hepa1-6 were transfected with both plasmids using the 4D-Nucleofector System (Lonza) using the EH-100 program in SF buffer. To select for cells expressing both gRNAs, GFP and mCherry double-positive cells were sorted 48 hr post transfection as single-cell deposition into 96-well plates using a FACS Aria (SORP) Cell Sorter (BD). Each single-cell clone was propagated and analyzed by genomic PCR and qRT-PCR to select for homozygous knockout clones. Cells transfected with a sgRNA targeting Renilla luciferase were used as a negative control.

MSCV-IRES-GFP-Pax3-FOXO1 (MSCV-P3F) and MSCV-IRES-GFP (MSCV-GFP) plasmids were a kind gift from Dr. Gerard Grosveld (St. Jude Children’s Research Hospital, Memphis). MiRZip-486-5p (MZIP486-5p-PA-1), MiRzip-scrm (MZIP000-PA-1 pGreenPuro Scramble Hairpin Control), MMIR-486 (MMIR-486-PA-1-microRNA Expression Construct) and MMIR-scrm (MMIR-000-PA-1 Mouse precursor Scramble negative control) were purchased from System Biosciences (USA). 293T were transfected with MSCV-P3F or MSCV-GFP using calcium phosphate. Viral supernatants were harvested at 48 h and 72 h. Virus particles were packaged using pPACKH1 HIV Lentivector Kit-LV500A-1 (System Biosciences, USA). Cells were transduced in suspension at 32 °C, 1250 × g for 1 h with 8 μg/ml Polybrene (hexadimethrine bromide; Sigma), and sorted using FACS Aria SORP cell sorter (BD) after selection with 2 μg/ml Puromycin (Abcam). For miRNA silencing, cells were transfected with 40 pmol of anti-mir-486-5p inhibitor (GenePharma, China) using Lipofectamine RNAiMAX (Invitrogen).