Brains from 40 adult naïve C57BL/6 mice were extracted, pooled, dissociated using Accutase, and purified using 40% Percoll as described above. Cells were counted, and an aliquot was removed for control single stains and FMO tubes. Fc receptors were blocked, and live cells were labeled with Calcein Violet AM as before. Cells were then stained with a cocktail of anti-A2B5, PDGFRα, NG2, O4, GALC, and MOG in flow cytometry buffer for 30 min at 4°C. Cells were washed twice and resuspended in flow cytometry buffer. Early OPCs (A2B5+PDGFRα+), intermediate OPCs (O4+NG2+), and mature oligodendrocytes (GALC+MOG+) were sorted by FACS into lysis/binding buffer (Miltenyi Biotec) using a FACSAria 5 (BD Biosciences). Sorted cells were stored at −80°C. After thawing on ice, total RNA was isolated using µMACS mRNA Isolation Kit (Miltenyi Biotec) and cDNA was synthesized on column with µMACS One-step cDNA Synthesis Kit (Miltenyi Biotec). Gene expression was analyzed by qRT-PCR using specific primers (Qiagen, Valencia, CA, Table S2
Facsaria 5
Manufactured by BD
The FACSAria 5 is a high-performance flow cytometer designed for cell sorting and analysis. It features advanced optics, fluidics, and electronics to enable precise cell identification and sorting. The FACSAria 5 provides reliable and reproducible results for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Streptomycin, by Corning (2 mentions)
Penicillin, by Corning (2 mentions)
Iq5 real time pcr detection system, by Bio-Rad (1 mentions)
Rt2 sybr green fast mastermix, by Qiagen (1 mentions)
Specific primers, by Qiagen (1 mentions)
Macs mrna isolation kit, by Miltenyi Biotec (1 mentions)
Lysis binding buffer, by Miltenyi Biotec (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Effectene, by Qiagen (1 mentions)
Cos 7, by American Type Culture Collection (1 mentions)
Moflo legacy, by BD (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dmso control, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Wortmannin w1628, by Merck Group (1 mentions)
Dispase, by STEMCELL (1 mentions)
6 protocols using facsaria 5
1
Oligodendrocyte Lineage Progression Analysis
2
Stable Cell Line Generation Protocols
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HEK293T, MDCK, and COS-7 cells were obtained from the American Type Culture Collection. A431D cells were provided by Sergey Troyanovsky (Northwestern University, Chicago, IL). All cell types listed were maintained in DMEM (Corning, New York, NY) containing 10% fetal bovine serum (FBS; Atlanta Biologicals or JRS Scientific, Woodland, CA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Corning). To generate the myr-E-cad cyto HEK293T stable line, cells were transfected with the myristoylated human E-cadherin cytoplasmic domain plasmid and the hygromycin resistance vector pCB7 (Michael Roth, UT Southwestern, Dallas, TX) in a 1:10 ratio using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cells were selected with 0.25 mg/ml hygromycin B (Calbiochem), and drug-resistant colonies were pooled. To generate E-cadherin-Dendra2 lines, cells were transfected with E-cadherin-Dendra2 using Effectene (Qiagen, Hilden, Germany) and selected with 800 μg/ml G418 (Corning). Resistant colonies were pooled and sorted for Dendra2 fluorescence by flow cytometry using a MoFlo Legacy (BD Biosciences) or FacsAria5 (BD Biosciences, San Jose, CA). IL2R-E-cadherin lines were generated by transfection of IL2R-E-cadherin with the G418 resistance vector pcDNA3 in a ratio of 1:10 using Effectene, selection with 800 μg/ml G418, and pooling of resistant colonies.
3
Multiparameter PBMC Isolation and Sorting
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From the S4 cohort, ∼2-3x108 PBMCs were separated into CD3+ and CD3- populations using magnetic beads (Figure 1 ). The CD3+ fraction was then split into two equally sized aliquots for T cell staining with either antibody panel 1 or 2. The CD3- fraction was also split into two aliquots: one aliquot (60%) for B cell and progenitor-cell staining with panel 3, and one aliquot (40%) for monocyte, DCs, NK cells and LD granulocyte staining with panel 4. After staining, the immune cells were sorted using a BD Influx for panel 1 and 3, a FACS Aria 5 for panel 2, and a FACS Aria 4 for panel 4 (all BD Biosciences). All cells were stained and sorted within 7 h after blood collection and kept on ice between processing steps. Sorting was performed to > 98% purity and then cells were lysed in TRIzol® reagent (Thermo Fisher Scientific) and stored at −80°C.
4
Regulated Cell Adhesion Disruption
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MDCK, A431D, and DLD1 cells (American Type Culture Collection) and DLD1 R2/7 αCat-deficient colon carcinoma cells (a gift from F. van Roy, Ghent University, Ghent, Belgium) were maintained in DMEM (Corning), containing 10% FBS (Atlanta Biologicals or JRS Scientific), 100 U/ml penicillin, and 100 µg/ml streptomycin (Corning). iDimerize cell lines were sorted for positive selection of mCherry fluorescence by flow cytometry using FACSAria 5 (BD Biosciences). mCherry FLαCat or FLαCatKKR<3A cell lines were selected in 5 µg/ml puromycin (Sigma-Aldrich) and were subsequently sorted for mCherry expression using flow cytometry. In drug treatment assays, cells were treated with one of the following: DMSO control (Sigma-Aldrich) or 1–20 µM wortmannin (W1628; Sigma-Aldrich) for 3 h. In forced-dimerization assays, cells were treated with 25, 250, or 500 nM B/B homodimer (635059; Clontech) for 20 min to 15 h, which was sometimes followed by treatment with 1 µM washout ligand (635088; Clontech).
5
Retroviral Transduction of γδ T Cells
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We cloned full-length cDNA of TCRγ and TCRδ chains, separated by self-cleaving T2A sequence, of either the 9C2 TCR28 (link) or GD8 TCR into the MSGV retroviral expression vector (Addgene #107226). CD8+ T cells were retrovirally transduced with GD8, 9C2, or FMC63-28Z anti-CD19 CAR as previously described73 (link) and sorted on a BD FACSARIA 5 (BD Bioscience) for γδTCR+ cells one day post transduction (Supplementary Fig. 6a ). After two days of expansion following sorting, γδ TCR and CD1d-PBS-57 staining was assessed by flow cytometry.
6
Isolation and Single-Cell Analysis of γδ T Cells
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Fresh tumor biopsy obtained from one PCGDTL patient (GD42) was obtained. Tissue was washed in PBS and digested overnight in a shaker in 60% RPMI and 40% dispase (StemCell Technologies). Tissue was washed in RPMI and filtered through a 70 μM filter. Cells were analyzed by flow cytometry for CD3, γδTCR, Vδ1, Vδ2, Vγ9 and live/dead staining. Live, CD3+, γδTCR+ were sorted using a BD FACSAria 5 and scRNA-seq performed via 10× Genomics single cell V(D)J analysis per the manufacturer’s instructions. To amplify γδ TCR transcripts, outer and inner enrichment primers targeting TRD and TRG gene constant regions (sequences listed below) were designed similar to the approach to αβ TCR transcript amplification, and all steps were performed per manufacturer instructions with the modification of replacing αβ TCR primers with γδ TCR primers, listed below.
TRD outer: 5′-GCTTGACAGCATTGTACTTCC-3′
TRD inner: 5′-GACAAAAACGGATGGTTTGG-3′
TRG outer: 5′-CATGTATGTGTCGTTAGTCTTCATG-3′
TRG inner: 5′-AGGAAGAAAAATAGTGGGCTTG-3′
TRD outer: 5′-GCTTGACAGCATTGTACTTCC-3′
TRD inner: 5′-GACAAAAACGGATGGTTTGG-3′
TRG outer: 5′-CATGTATGTGTCGTTAGTCTTCATG-3′
TRG inner: 5′-AGGAAGAAAAATAGTGGGCTTG-3′
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