Lentiviral vector FUW-Luc-mCherry-puro lentivirus (FmC) used in this study, encoding Firefly luciferase and mCherry (from Dr. Andrew Kung, Columbia University) was packaged in 293T cells using a helper virus-free packaging system. Optimal conditions for successful luciferization were established individually for each PDX model (Supplemental Table 1 ). In general, ascites from established PDX models were implanted intraperitoneally in NOD-SCID IL2Rγnull mice (NSG, Jackson Laboratory) after a comparative DF14-Luc tumor growth rate study demonstrated that latency and growth rates were superior in NSG mice, as compared to SCID or irradiated nude mice (data not shown). Fresh ascites-derived tumor cells from these PDX tumor-bearing NSG mice were then plated ex vivo. They were transduced with FmC Lentiviral vector at a multiplicity of infection of ~10 in medium containing polybrene at 8 µg/ml and selected in puromycin-containing media for 5 to 7 days. The selected cells, once confirmed to be expressing RFP by fluorescent microscopy (Leica) were directly injected into NSG mice intraperitoneally and further expanded (Supplemental Figure 1 , Schema). Luciferized PDX models were then further expanded (to a maximum of six passages), banked, characterized, and utilized for drug efficacy and biomarker evaluation studies.
Fluorescent microscopy
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Fluorescent microscopy is an imaging technique that uses fluorescent molecules or proteins to visualize and analyze the structure and function of cells, tissues, or other biological samples. The core function of a fluorescent microscope is to excite fluorescent labels within the specimen and capture the emitted light, providing high-contrast images that can reveal intricate details and specific targets within the sample.
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Lab products found in correlation
Cy3 conjugated anti rabbit igg, by Abcam (4 mentions)
Image pro plus, by Leica (4 mentions)
Image pro plus 6, by Media Cybernetics (4 mentions)
Fitc conjugated anti mouse igg, by Abcam (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Autophagy related proteins, by Cell Signaling Technology (2 mentions)
Rabbit anti vegf c, by Novus Biologicals (2 mentions)
Vegf c elisa, by Novus Biologicals (2 mentions)
Rat anti vegfr3, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Nod scid il2rγ null mice, by Jackson ImmunoResearch (1 mentions)
Transwell insert, by Corning (1 mentions)
Ldh release assay, by Roche (1 mentions)
Tagbfp, by Evrogen (1 mentions)
Edu labeling kit, by RiboBio (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Rabbit anti s100β, by Abcam (1 mentions)
65 protocols using fluorescent microscopy
1
Lentiviral Transduction of PDX Models
2
Evaluating Cell Proliferation Assays
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A CCK-8 kit was used to assess the proliferation of cells at specific time points based on provided instructions. Briefly, cells were seeded in 96-well plates (2 × 103/well), followed by the addition of CCK-8 solution to each well and incubation for 15 h. Absorbance at 450 nm was then assessed with a spectrophotometer. Colony formation assays were conducted by plating cells in 6-well plates (800/well) and incubating them for 14 days, followed by fixation with 4% paraformaldehyde (PFA) and staining with 0.5% crystal violet. EdU uptake was assessed as reported previously [11 (link)]. Briefly, cells were added to 96-well plates (1 × 103/well) for 24 h, after which EdU (50 μM) was added, and cells were fixed for 15 min with 4% formaldehyde prior to permeabilization for 20 min using 0.5% Triton X-100. After rinsing with PBS, 100 μL of 1× Apollo reaction cocktail was added, cells were incubated for an additional 30 min, and they were then imaged via fluorescent microscopy (Leica, Germany).
3
BMSC-MM Coculture Enhances MO Migration
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When BMSCs reached 70% confluence, they were directly cocultured with MM cells (2 × 105 cells per well) in 24-well plates. CFSE-labeled MOs were put in transwell inserts (pore size: 5 μm, Corning) and cocultured with MM/BMSC or primary MM BM cells overnight. MOs migrating to the lower chamber were detected by fluorescent microscopy (50 ×, Leica) for CFSE+ cells. MOs migration to the lower chamber in medium only was the control. For quantification of the results, migrated cells in three randomly chosen view zones were counted and the mean value was used for plotting. In some experiments, chemokine neutralizing antibody or control IgG were added in the coculture (a final concentration of 10 μg/ml) to neutralize the chemokines.
4
Cortical Neuron NMDA Excitotoxicity Assay
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Since a larger quantity of cortical neurons can be obtained from one embryo compared to hippocampal neurons, cortical neurons were used for NMDA excitotoxicity assay. Cortical neurons at 11–12 days in vitro (DIV) were pre-treated with PSR or vehicle (DMSO) for 2 h and subsequently rinsed with Locke’s solution (5 mM KCl, 128 mM NaCl, 2.7 mM CaCl2, 1 mM Na2HPO4, 5 mM HEPES, and 10 mM glucose) followed by a 15-min incubation with glycine-containing (10 μM) Locke’s solution. The neurons were then co-treated with PSR (or vehicle) and 20 μM NMDA dissolved in Locke’s plus glycine solution for 20 min. The neurons were then incubated with fresh growth medium. After 24 h, NMDA excitotoxicity was assessed by either the lactate dehydrogenase (LDH) release assay (Roche) or immunohistochemistry, as described previously [52 (link)]. The cell death in the presence of PSR and NMDA was quantified by normalizing the LDH release to the maximum (vehicle with NMDA, the control) and minimum (no NMDA) LDH releases. Neurons were immunostained with β-tubulin type III antibody (1:1000, Sigma) and labeled with FITC-conjugated secondary antibody. DAPI (4′, 6-diamidino-2-phenylindole) was used to stain the nuclei. Images were captured and visualized by fluorescent microscopy (Leica).
5
Automated Immunofluorescence Imaging Protocol
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Immunofluorescence analysis was performed according to protocols. Cells were implanted in 24-well dishes and fixed by 4% paraformaldehyde 24 h later. Fixed cells were stained with autophagy-related proteins (Cell Signaling Technology, USA), Rage (Cell Signaling Technology), followed by FITC-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG (Abcam). Representative images were detected by fluorescent microscopy (Leica, German) and data were processed via ImagePro Plus.
6
Generating Lentiviral Vector p53dd
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The pLV-S16-p53dd vector expressing p53dd [34 (link)] was generated by cloning human p53dd (p53 aa 1–11,305–393) in frame behind Tag-BFP (Evrogen, Cat#26291) and cloning of the resulting fusion ORF downstream of the CMV promoter in the p156RRLsin backbone [35 (link)]. The 3rd generation pLV-S16-p53dd lentiviral vector was packaged by co-transfection with Lipofectamine 2000 (ThermoFisher) into HEK293T cells with the packaging plasmids pMD2.G, pRRE and pRSV/REV. Fresh medium was added 6 h after transfection and collected 48h later to be ultra-centrifuged at 24,000 rpm 4C for 2h. The pellet was re-suspended in DMEM-10% FBS and used to infect caPSC-82. Successful infection was monitored by visualization of the BFP using fluorescent microscopy (Leica). Over time, p53dd-expressing cells out-grew non-infected cells and all cells were identified as BFP-positive.
7
Immunofluorescence Staining of Cells
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Immunofluorescence was performed as previously described21 (link). Briefly, cells were seeded in 24-well dishes and fixed by 4% paraformaldehyde and stained with primary antibodies followed by FITC-conjugated secondary antibodies. Images were collected by fluorescent microscopy (Leica, Germany) and processed with ImagePro Plus.
8
EdU Labeling for Cell Proliferation
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EdU labeling medium was added to cell culture with EdU labeling kit (RiboBio, Guangzhou, China). HeLa or SiHa cells after transfection were incubated for 2 h, and then fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Cells were washed at room temperature by PBS (Sigma-Aldrich) dyed with anti-EdU (Acbam, Cambridge, USA) working solution (Invitrogen). Under the same conditions, cells were again washed with Triton X-100 (Solarbio, Shanghai, China) in PBS (Sigma-Aldrich). Cells were observed via a fluorescent microscopy (Leica, Wetzlar, Germany).
9
Immunofluorescence Analysis of NF-κB Pathway
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Cells were plated in 96-well culture plate and after 45 min of TNF-α treatment were fixed in 4% paraformaldehyde. Cells were washed, incubated with 0.1% PBS-Triton, blocked with 0.5% BSA in PBS and incubated overnight at 4 °C with a 1:150 dilution of the primary antibodies against human ERα, RelA, RelB, cRel, p100/p52, p105/p50 (Cell Signaling Technology, Europe, B.V). Then, cells were washed 3 times and incubated with the secondary antibody FITC-conjugated anti-rabbit diluted 1:200. Nucleus cells were stained with a DAPI (4′,6-diamidino-2-phenylindole) solution and pictures were taken with fluorescent microscopy (Leica). As an internal control, cells were incubated with a secondary antibody without prior incubation with the primary antibody.
10
Quantification of Apoptosis in KBv Cells
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The cell apoptosis activity was detected by the assessment of nuclear morphology in KBv cells by Hoechst 33342 staining. Briefly, cells were seeded in six-well plates containing a coverslip with 5×105 cells per well and cultured for 24 hours. Cells were then incubated for another 24 hours with a mixture of DOX and PTX (DOX+PTX, DOX:PTX =2:3, w/w), c(RGDyK)-FP-DP, or PF-DP (total drug concentration of 1 μg/mL), and DMEM was used as the control group. After incubation, the medium was removed and cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) at room temperature for 15 minutes, stained with 10 μg/mL Hoechst 33342 in PBS at room temperature for 15 minutes, and then washed twice with ice-cold PBS followed by the observation using fluorescent microscopy (Leica).
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