OT1 CD8+ T cells were isolated as described above and incubated with unconjugated, Db-GP33, or Kb-OVA liposomes at 1 μg/ml in T cell media for 24 hr. T cells were washed and then co-incubated with Renilla luciferase expressing B16-OVA cells (~50–60% confluency) at 1:1, 5:1, or 10:1 effector-to-target cell ratios in T cell media for 24 hr. Luciferase activity was measured by adding ViviRen™ In Vivo Renilla Luciferase Substrate (Promega) according to the manufacturer’s instructions and collecting on a Cytation 5 plate reader (BioTek).
Viviren in vivo renilla luciferase substrate
Manufactured by Promega
Sourced in Switzerland
ViviRen™ In Vivo Renilla Luciferase Substrate is a non-invasive, bioluminescent substrate used for the detection and quantification of Renilla luciferase reporter gene expression in living cells and animal models.
Automatically generated - may contain errors
5 protocols using viviren in vivo renilla luciferase substrate
1
CD8+ T Cell Cytotoxicity Assay
2
Bioluminescent Imaging of Luciferase Activity
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To detect Fluc activity, we dissolved D-luciferin (Promega) in PBS at 15 mg/mL and administered intraperitoneally at a dose of 100 μL (150 μg) per animal. To detect Renilla luciferase (Rluc) activity, we dissolved ViviRen in vivo Renilla luciferase substrate (Promega) in dimethyl sulfoxide at 10 mg/mL, and the stock solution was further diluted 1,000-fold in PBS containing 0.1% bovine serum albumin, after which 100 μL (1 μg) was administered by intravenous injection. The mice were anesthetized with isoflurane before imaging with the in vivo imaging system (IVIS) 100 bioluminescence imaging system (Xenogen). Bioluminescence signals were quantified 10 min after the injection of D-luciferin or immediately after the injection of ViviRen according to the manufacturer’s protocol.
3
Evaluating CALB2 Knockdown in NSG Xenograft Model
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NOD SCID gamma (NSG) mice were utilized (4 mice per group). All experiments were performed with permission of the local animal care committee (Canton of Fribourg, Switzerland) and according to the present Swiss law and the European Communities Council Directive of 24 November 1986 (86/609/EEC). Briefly, MSTO-211H-Rluc cells were transduced for 24 h with a lentiviral vector containing either an shRNA against GFP (control group) or against CALB2 (test group) at a MOI of 10. Mice were separated into two groups and injected with cells pre-treated with shGFP or with shCALB2. Direct intraperitoneal (i.p.) injection of 1.5x106 tumor cells in 200 μl PBS serum-free media was performed and mice were scanned at days 16 and 30 post-injection in order to follow tumor progression with the IVIS Lumina II In Vivo Imaging System (Caliper Life Sciences, Hopkinton, USA). For the Renilla luciferase detection imaging, 1 mg/kg ViviRen™ In Vivo Renilla Luciferase Substrate (Cat#P1231; Promega, Dübendorf, Switzerland) was injected i.p. before imaging. Images were acquired for 5-30 seconds depending on signal strength. Luminescence of the tumor was quantitatively evaluated using the Living Image 4.2 Software and the BLI signal was reported as photons/s/cm2/sr. Organ samples were collected (diaphragm and parietal layer) for histological analysis.
4
In Vivo Bioluminescence Imaging of Murine Myeloma
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Murine AB12 MM cells were transduced with lentivirus encoding pLV-hRluc resulting in stable expression of the reporter Renilla Luciferase [38 (link)]. BLI was performed in mice injected intraperitoneally with 1x105 AB12-LV-hRluc cells using the IVIS Lumina II In Vivo Imaging System (Caliper Life Sciences, Hopkinton, USA). Directly before BLI each mouse received an intraperitoneal injection (1 mg/kg) of luciferase substrate ViviRen™ In Vivo Renilla Luciferase Substrate (Cat#P1231; Promega, Dübendorf, Switzerland) following the manufacturer’s instructions. Two time points were chosen for BLI, i.e. 48 h after AB12-LV-hRluc cell injections (before treatment) and 144 h after cell injection (4 days FCF treatment). For the treatment, FCF was dissolved in propylene glycol (PG) to 25 mg/ml and 100 µl (or 100 µl PBS control) was injected intraperitoneally (n=4 per group). For BLI, mice were kept at 37ºC and under 2-3% isoflurane/l/min O2 anesthesia during image acquisition and FCF injection. Images were acquired for 5-30 s depending on signal strength. The flux (photons/seconds) in a Region of Interest (ROI) placed over the peritoneal region of the mice was used to quantify BLI signals on the acquired images using the Living Image 4.2 Software. Values of BLI signals are expressed as photons/s/cm2/sr.
5
In Vivo Renilla Luciferase Imaging in BALB/c Mice
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BALB/c mice were infected with rOC43-ns2Del-Rluc and treated with lycorine or DMSO-PBS, followed by immediate anesthetization with 2% isoflurane and intraperitoneal administration of ViviRen in vivo Renilla luciferase substrate (20 μg/g; Promega) at 2, 3, and 4 days postinoculation. The mice were positioned in a specially designed box and placed onto the stage inside the light-tight camera box. Mice were imaged within 5 min postinjection of the substrate, and photon flux was quantitated using Living Image software (PerkinElmer, Waltham, MA, USA).
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