hiPSCs were differentiated to astrocytes based on our recent protocol [10 (link)]. Fetal human cortical astrocytes were obtained from ScienCell (#1800). Cells were cultured according to suppliers’ instructions and used at passage <8 post-thawing. One million cells were thawed in complete astrocyte media (AM; ScienCell 1801). For assays, 500,000 cells/well were seeded in 6-well cell culture plates coated with 1x attachment factor (AF; Thermo S-006-100). On the next day of subculture (day 1), the media were changed to a 1:1 mixture of AM and “defined media,” and on day 2 to defined media only. Our defined media were composed of DMEM without glucose and glutamine (Thermo A14430-01) supplemented with 1% G-5 (Thermo 17503012; serum-free supplement for growth and expression of glial cells), 1% Penicillin-Streptomycin (P/S; ScienCell 0503), 10mM GlutaMax (Sigma 35050-38), 25 mM HEPES (EMD 391340), and 5.55 mM glucose (Sigma G7528) to match the AM glucose content. We used these defined media to have better control over the initial concentration of glucose within the media. Exemplary bright-field images of the cells on day 2 are shown in Figure 1 b,c.
S 006 100
Manufactured by Thermo Fisher Scientific
Sourced in United States
The S-006-100 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed for general laboratory use. The core function of this product is to perform specific tasks in a laboratory setting. No further details can be provided in an unbiased and factual manner without speculating on its intended use.
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Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Beas 2b, by Thermo Fisher Scientific (2 mentions)
Tr 1003 g, by Merck Group (2 mentions)
Penicillin streptomycin p s, by ScienCell (1 mentions)
Glutamax, by Merck Group (1 mentions)
A1443001, by Thermo Fisher Scientific (1 mentions)
Crl 2128, by American Type Culture Collection (1 mentions)
Nu serum, by Corning (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Cell proliferation dye efluor 670, by Thermo Fisher Scientific (1 mentions)
Calcein green, by Thermo Fisher Scientific (1 mentions)
Phc3016, by Thermo Fisher Scientific (1 mentions)
Accuri c6 cytometer, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Beas 2b, by American Type Culture Collection (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Hek293, by Thermo Fisher Scientific (1 mentions)
Bronchial epithelial growth medium (begm), by Thermo Fisher Scientific (1 mentions)
Singlequots supplement pack, by Lonza (1 mentions)
7 protocols using s 006 100
1
Astrocyte Differentiation and Culture
2
Cyclic Stretch Induces Vascular Smooth Muscle Cell Response
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Cyclic stretch was performed using a uniaxial cell stretch system (Central Workshop Tsukuba University) as described in a previous study (Yamashiro et al., 2020 (link)). We employed rat vascular smooth muscle cells (SMCs) or U2OS cells. The cells were plated on silicon elastomer bottomed culture plates (SC4Ha, Menicon Life Science) coated with a cell attachment factor containing gelatin (Thermo Fisher Scientific, S006100) and subjected to cyclic stretch with a frequency of 1.0 Hz (60 cycles/min) and 20% strain for 6 h.
3
Androgen Production in H295R Cells
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Human adrenocortical-carcinoma cells (H295R cells) were used as an in vitro cell culture model for androgen production. These cells are commonly used in studies of steroidogenesis and androgen biosynthesis pathways [21 (link)–23 (link)]. H295R cells were purchased from ATCC (Manassas, VA, USA, cat. no. ATCC® CRL-2128™) and cultured per the recommended guidelines. Briefly, H295R cells were cultured in flasks pre-coated with extracellular matrix (Gibco, USA, cat. no. S-006-100) with DMEM/F12 (Gibco, cat. no. 21041025) and 2.5% Nu-Serum (Corning, USA). The cells were subcultured at a ratio of 1:3 to 1:4 and culture media were changed twice a week.
4
Transwell Assay for CAR T Cell Migration
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5µm-pore transwell inserts (Costar 3421) have been coated for 1 h at 37 °C with gelatin (Gibco S006100) and 10 µg/ml bovine fibronectin protein (Thermo Fisher 33010018). Subsequently, 5 × 105 HUVEC cells were added into the insert, let expanded for 24 h and stimulated o.n. with 10 ng/ml human TNF (Thermo Fisher PHC3016). The day after, CAR T cells were stained with either Calcein Green or Cell Proliferation Dye eFluor670 (Thermo Fisher 65-0840-85) (same procedure used for motility assay, see above) and mixed in a 1:1 ratio. Then, 2 × 105 cells were added to the transwell upper chamber in a motility medium (BSA, no FBS). Transwell lower chamber was filled with RPMI complete medium (10% FBS). The transmigrated cells were collected and quantified by BD Accuri C6 cytometer.
5
Human Cell Line Culture Protocols
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Human embryonic kidney cells (HEK293) and human bronchial epithelial cells (BEAS-2B) were purchased from ATCC (Manassas, VA, USA). HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Gibco, ThermoFisher, Waltham, MA, USA), 10 U penicillin/mL, and 10 g streptomycin/mL (ThermoFisher Scientific, Waltham, MA, USA). Cells were maintained in 10 cm tissue culture plates at 37 °C under 5% CO2. BEAS-2B cells were cultured in either DMEM (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Gibco, ThermoFisher) for the short term treatments, or 1× Bronchial Epithelial Cell Growth Medium (BEGM) (Lonza, Basel, Switzerland) SingleQuots Supplement Pack (Lonza, Basel, Switzerland) containing 2 mL BPE, 0.5 mL Insulin, 0.50 mL Hydrocortisone, 0.5 mL GA-1000, 0.5 mL Retinoic Acid, 0.5 mL Transferrin, 0.5mL Triiodothyronine, 0.5 mL Epinephrine, and 0.5 mL hEGF for the long term treatments. Cells were maintained in 6-well tissue culture plates at 37 °C under 5% CO2. The BEAS-2B cells cultured for 9 weeks in BEGM were maintained in 6-well tissue culture plates coated with a 2:1 mixture of 0.1% gelatin (s006100, Gibco, ThermoFisher) and 0.01 mg/mL (Gibco, ThermoFisher).
6
Transduction of hiF-T Cells for Single-Cell RNA-Seq
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To transduce hiF-T cells, we freshly thawed virus-laden media on ice, added it to dissociated cells with 4ug/mL of polybrene (Millipore-Sigma #TR-1003-G), and plated 50,000 cells/well in a 6-well plate coated with Attachment Factor (Fisher #S006100). The volume of virus-laden media used was decided by measuring the multiplicity of infection (MOI) with different viral titers. For single-cell RNA-sequencing experiments, we aimed for a low MOI with 10%−25% GFP-positive cells to minimize the fraction of cells with multiple unique barcodes. We found it relatively computationally challenging to differentiate multiple-barcoded cells from doublets introduced by gel beads-in-emulsions. This was not an issue for bulk DNA clone barcode overlap experiments for which we aimed for a high MOI with 60–70% GFP-positive cells. We used 35 uL/well of virus-laden media for low MOI and 80 uL/well for high MOI. After plating hiF-T cells with virus, we performed a 30 min incubation at room temperature before centrifuging the 6-well plate at 930g for 30 min at room temperature. After 24 hours, we passaged the cells from 2 wells onto 10 cm plates or from 6 wells onto 15cm plates. The barcoded cells (GFP-positive) were sorted for all single-cell RNA-sequencing experiments but not for bulk DNA clone barcode overlap experiments.
7
Lentiviral Transduction of hiF-T Cells
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To transduce hiF-T cells, we freshly thawed virus-laden media on ice, added it to dissociated cells with 4 ug/mL of polybrene (Millipore-Sigma #TR-1003-G), and plated 50,000 cells/well in a 6-well plate coated with Attachment Factor (Fisher #S006100). We used 400 uL/well of virus-laden media, aiming for a high MOI. After plating hiF-T cells with virus, we performed a 30 min incubation at room temperature before centrifuging the 6-well plate at 930g for 30 min at room temperature. After 24 hours, we passaged the cells from 2 wells onto 10 cm plates and began selection in 2.5 ug/mL of blasticidin. This selection was removed after 7 days, which corresponded with when cell death was near complete in control hiF-Ts we cultured in parallel containing no lentiviral constructs. We wanted to minimize the effect of blasticidin on reprogramming, so we cultured the cells for 7 days without blasticidin and without puromycin before reprogramming via OKSM induction.
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