Plv mcherry

Keratinocytes were seeded on collagen-coated PDMS substrates 24 h prior to live imaging. In some experiments, keratinocytes were infected with lentiviral vectors encoding GFP (pLenti-CMV GFP puro (658-5), gift from Eric Campeau, Addgene plasmid # 17448,) and mCherry (pLV-mCherry, gift from Pantelis Tsoulfas, Addgene plasmid # 36084). Keratinocytes were transduced with lentiviral plasmids carrying GFP and mCherry reporters 2 days prior to live imaging. A JuLI™ Stage Real-Time Cell History Recorder device was used to acquire images from live keratinocytes. Images were captured with an interval time of 1–2 h and videos were created using JuLI™ Stage edit software.

Isolated LEC and BEC populations were retrovirally infected with fluorescent proteins to visualize network formation. The cDNA encoding pLV-EGFP, pLV-mCherry, and pBMN-Z was purchased from Addgene (Cambridge, MA, USA). The pcDNA3-EYFP-HIS was purchased from Invitrogen (ThermoFisher, Waltham, MA, USA). The eGFP and mCherry were subcloned into the pBMN backbone after digestion with BamHI and SalI. The EYFP-HIS was subcloned into pBMN after digestion with BamHI and EcoRI. Phoenix Ampho cells were a kind gift from Regina Grillari (University of Natural Resources and Life Sciences, Vienna, Austria) and cultured in DMEM 10% FCS. Virus particle generation was performed by transfecting Phoenix cells at 80% confluency using Lipofectamine 2000 or TurboFect (ThermoFisher, Waltham, MA, USA), following the manufacturer’s instructions. The supernatants containing virus particles were transferred onto 80% confluent LEC and BEC, and virus particles were centrifuged onto the cells at 800 × g for 60 min. The supernatant was then removed, and the cells were incubated overnight in EGM-2. Retrovirally infected cells were then expanded in new flasks and used for subsequent experiments.

The plasmid pLV-mCherry (catalog no.36084, Addgene) was cotransfected with the packaging plasmids pCMV Δ R8.2 (catalog no.12263, Addgene) and pMD2.G (catalog no.12259, Addgene) into 293T cells to package lentivirus Lenti-mCherry. The plasmid pCDH-3xFLAG-GFP-puroR (catalog no.167463, Addgene) was inserted into cloned mouse polyomavirus middle T antigen transcripts before cotransfection with pMDLg/pRRE (catalog no.12251, Addgene) and pRSV-Rev (catalog no.12253, Addgene) in 293T cells to package Lenti-MT. The viral supernatant was harvested at 48 and 72 hours after transfection. After centrifuging at approximately 500g for 5 minutes to pellet any packaging cells and filtering through a 0.45 μm PES filter, the virus supernatant was used for the transduction of target cells. DMSO3-1-mCherry cells was selected using fluorescence-based screening, while melanoma-MT cells were selected using puromycin selection. The cells were subsequently screened to confirm the expression of the target protein.

SD2 GSCs were labeled with mCherry expressing lentivirus (pLV-mCherry; Addgene plasmid 36084) and then cultured in 3D fibrin gel (10 mg/ml fibrinogen +3 U/ml thrombin; Sigma-Aldrich), and images were taken after 27 days of culture. Primary human brain microvascular endothelial cells (bEC; Cell Systems) expressing EGFP were first cultured on laminin-coated tissue culture flasks with endothelial cell growth medium 2 (EGM2; Promocell). For bEC spheroid formation, bEC were cultured in hanging drop condition with 0.0625% Methylcellulose (Sigma) in media. Each hanging drop containing 1000 endothelial cells was cultured for 24–36 h. Human astrocytes and brain vascular pericytes were cultured with astrocyte medium and pericyte medium (cells and media from Sciencell). To create a 3D brain microenvironment to assay GSC invasion, three cell types (astrocytes, pericytes, and bEC spheroids) were embedded in the fibrin matrix (10 mg/ml fibrinogen +3 U/ml thrombin; Sigma-Aldrich) along with sparsely distributed GSC. Seeding densities of cells were 3 million cells/ml for astrocytes and pericytes, 6 million cells/ml for bECs, and 25,000 cells/ml for GSCs. The 3D constructs with four cell types were cultured in EGM2 medium for 14 days and then imaged using fluorescence microscopy (Nikon Eclipse Ti2).

The following constructs, LVs and AAVs were used: LV.PGK.GFP (pRRLSIN.cPPT.PGK-GFP.WPRE plasmid #12252 from Addgene), LV.CMV.mCherry (pLV-mCherry plasmid #36084 from Addgene), LV.PGK.iGluSnFR (pCMV(MinDis).iGluSnFR plasmid #41732 from Addgene cloned into pRRLSIN.cPPT.PGK-GFP.WPRE plasmid #12252 by replacing GFP with iGluSnFR), AAV5.SYN.GCaMP6f (#AV-5-PV2822 from U Penn Vector Core facility), LV.PGK.BDNF-mCh^{36 (link)} and pDsRed2-Mito (plasmid #632421 from Clontech).