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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
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  • Dyskusje
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  • Materiały
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Podsumowanie

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

Streszczenie

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 µL of poly-L-lysine (PLL) is slowly instilled at a rate of 10 µL/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 µL) at a rate of 10 µL/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.

This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

Wprowadzenie

The goal of this method is to generate murine orthotopic bladder tumors and to monitor the implanted tumors as accurately as possible, so that mice without tumor implantation are not thought to have been cured at end-point analysis. Overall, the method shown will reduce the need for large numbers of mice for experimental analysis and ensure greater accuracy in determining therapeutic outcomes.

The development of an orthotopic model for cancer is important, as implanting tumor cells subcutaneously does not recapitulate the environment of the clinical disease or enable the development of therapeutic strategies. The architecture of the bladder permits the instillation of bladder cancer therapies directly into the bladder with minimal systemic effects. Thus, animal models that recapitulate this environment, such as an orthotopic model, are important to evaluate new therapies. The conclusions drawn from any experimental set-up are dependent upon the limitations of the model.

Several techniques have been developed for the production of orthotopic bladder tumors in mice. These rely on damaging the glycosaminoglycan layer of the bladder, enabling tumor cells to be implanted. The techniques used include electrocautery, which results in a single point of damage in the bladder wall, leading to tumor development at one site in the bladder1,2. However, the success rate of tumor implantation using electrocautery is operator-dependent varying from 10 - 90%, and it includes the danger that the bladder wall will be punctured, leading to tumors developing in the peritoneal cavity. Chemical cautery is performed using silver nitrate, which damages the bladder wall3. Similarly, acid has been used to damage the bladder wall4. Trypsin has also been used to damage the bladder as well5. These methods may result in the development of more than one tumor in the bladder. Furthermore, there is a danger of severe damage to the bladder if the chemicals are left in contact with the bladder wall for too long. The method developed by Ninalga et al. uses the positively charged poly-L-lysine (PLL)6 molecules to coat the bladder wall; this enables the negatively charged tumor cells to stick to the glycosaminoglycan layer of the bladder. This method generally results in more than one tumor developing in the bladder, but tumor implantation is at 80 - 100%4,7. Technically, it is also the easiest procedure to perform. To ensure that the tumors that develop are fairly even in size, it is important that the tumor cells are not grouped in large clumps before implantation.

In order to evaluate therapeutic efficacy, it is best to perform this study on mice with fairly similar-sized tumors. Thus, a good detection system that can quantify tumor size soon after implantation is important. Several strategies have been used to evaluate tumors. These include magnetic resonance imaging (MRI)8-10, fluorescence11, bioluminescence12,13, ultrasound14, and enzyme-linked immunosorbent assay (ELISA)15,16. While MRI and ultrasound do not require modifications of the tumor cells, there is a need for sensitive equipment and contrast agents for MRI. The fluorescence-, luminescence-, and ELISA-based assays require modification of the tumor cells to express marker proteins that can be detected by these methods. For luminescence, a substrate is required for the detection of the luciferase activity; thus, there is an added step and increased cost. Both luminescence and fluorescence require specialized equipment. To produce fluorescence, green fluorescent protein (GFP) cyclization, which is catalyzed by molecular oxygen, is required. Thus, GFP expression may be variable within a tumor mass depending on access to oxygen, making this a rather unreliable marker17. Modification of the murine bladder cancer cell line MB49 to secrete human Prostate Specific Antigen (PSA)15,16 as a surrogate marker is another strategy. These markers also provide an alternative means of confirming tumor presence at the termination of the experiment, making them an alternative to immunohistochemistry. This study reports the PLL method of orthotopic tumor implantation and presents a comparison of tumor detection systems, namely ELISA, fluorescence, and ultrasound imaging.

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Protokół

All animal work adhered to the Institutional Animal Care and Use Committee (IACUC) guidelines on animal use and handling (Protocol number 084/12) at the National University of Singapore.

1. Growing MB49-PSA Cells In Vitro and Measuring PSA Secretion

  1. Maintain murine bladder cancer cells MB49-PSA15 in complete Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, and 0.05 mg/mL Penicillin-Streptomycin in an incubator at 37 °C and 5% CO2. Add 200 μg/mL Hygromycin B to maintain the selection pressure of PSA-secreting cells.
  2. To determine PSA secretion, plate 1 x 106 cells in a 6-well culture plate and incubate it at 37 °C in the presence of 5% CO2 for 48 h.
  3. Two days later, collect the supernatant for the PSA measurement.
    1. Using a Pasteur pipette, transfer the supernatant to a 15 mL centrifuge tube.
    2. Centrifuge for 5 min at 250 x g to remove floating cells and debris. If the PSA assay is carried out on a different day, store the supernatant at - 30 °C in a 1.5 mL microcentrifuge tube. If not, proceed to the next step immediately.
    3. Determine the PSA concentration using a human-free PSA ELISA kit7.
  4. Enumerate the plated cells.
    1. Using a Pasteur pipette, wash the cells gently with 1 mL of DMEM. Aspirate and completely remove the media.
    2. Add 1 mL of fresh media and scrape gently with a cell scraper to dislodge the cells from the well.
    3. Transfer the cells to a microcentrifuge tube and re-suspend them thoroughly to ensure a single-cell suspension.
    4. Mix equal volumes of the cell suspension and a 0.4% trypan blue solution. Count the live cells (which do not take up the dye) using a hemocytometer.
  5. Calculate the PSA expression as ng of PSA/mL of media/106 cells. The PSA expression of MB49-PSA cells for implantation in mice is 80 - 120 ng/mL/106 cells.

2. Determining the Sensitivity of PSA Measurements by ELISA and Real-time PCR Analysis

  1. Plate MB49-PSA cells in complete DMEM media in a 6-well culture plate with different amounts of parental MB49 cells such that there is a maximum of 1 x 106 cells per well (i.e., 100, 101, 102, 103, 104, 105, or 106 MB49-PSA cells are cultured with 106, 105, 104, 103, 102, 101, or 100 MB49 cells, respectively).
  2. One day later, collect the supernatant for PSA measurement, as described in step 1.3. Determine the PSA concentration using a human-free PSA ELISA kit7.
  3. Extract the RNA from the cells.
    1. Lyse the cells directly in the plate by adding 1 mL of RNA extraction reagent.
    2. Incubate for 5 min at room temperature and transfer the cell lysate to a microcentrifuge tube. Add 0.2 mL of chloroform and vortex the samples vigorously for 15 s. Incubate them for 3 min at room temperature.
    3. Centrifuge the samples at 12,000 x g for 15 min at 4 °C. Carefully transfer the upper aqueous phase (0.5 mL) to a fresh tube without disturbing the interphase.
    4. Add 0.5 mL of isopropyl alcohol. Incubate for 10 min at room temperature. Centrifuge the samples at 12,000 x g for 10 min at 4 °C. Remove the supernatant completely, without disturbing the RNA precipitate at the bottom of the tube.
    5. Wash the pellet once with 1 mL of 75% ethanol. Centrifuge at 7,500 x g for 5 min at 4 °C. Remove all ethanol and allow the pellet to air dry for 10 min.
    6. Dissolve the RNA in 30 µL of nuclease-free water. Incubate for 5 min at 60 °C to increase the solubility.
    7. Quantitate the RNA by measuring the absorbance using ultraviolet (UV) spectroscopy at 260 nm (A260). To assess RNA purity, measure the absorbance at 280 nm (A280) and calculate the A260/A280 ratio which should be above 1.6.
  4. Reverse-transcribe cDNA from 2 µg of RNA.
    1. Prepare the reaction mixture on ice and transfer it to 0.2 mL tubes.
    2. Briefly centrifuge the tubes to spin down the contents and to eliminate air bubbles.
    3. Load the tubes into the thermal cycler and perform reverse transcription at the following conditions: 60 min at 37 °C followed by 5 min at 95 °C. Once the run is completed, keep the cDNA samples at 4 °C.
    4. Store the samples at - 30 °C for long-term storage.
  5. Perform a real-time PCR analysis for PSA and cytoplasmic beta actin. Beta actin is used to normalize the samples. Perform the assay on 100 ng of reverse-transcribed RNA in a 96-well plate. Assay all samples in triplicate. Perform 40 cycles with the following parameters: 2 min at 50 °C, 10 min at 95 °C, 15 s at 95 °C for denaturation, and 1 min at 60 °C. Set the lowest limit of detection at a cycle threshold (CT) of 35; thus, any sample with a CT value beyond 35 is considered not detectable.

3. Maintaining the Tumorigenicity of the MB49-PSA Cell Line

NOTE: Prolonged growth in vitro leads to a loss of tumorigenicity. To maintain tumorigenicity, the MB49-PSA cell line is passaged through the mouse at least once every 2 years.

  1. Cells.
    1. Harvest exponentially growing cells by scraping them gently with a sterile cell scraper. Mix equal volumes of the cell suspension and a 0.4% trypan blue solution. Count the live cells using a hemocytometer. Do not implant if more than 20% of the cells are dead.
    2. Calculate the amount of live cells required (1 x 107 cells/mL) and transfer them to a 15 mL centrifuge tube. Centrifuge at 250 x g for 5 min at 4 °C and discard the supernatant. Re-suspend the cell pellet in DMEM blank media. Repeat the wash three times to remove all traces of FBS before implantation.
    3. Keep the cell suspension on ice until the mice are ready for implantation.
  2. Implant the tumor cells subcutaneously in mice on the dorsal flank.
    1. Anesthetize a 4- to 6-week-old C57BL6/J mouse using a mixture of the anesthetics ketamine and medetomidine (75 mg/kg and 1 mg/kg, respectively), injected intraperitoneally at 0.1 mL per 10 g of bodyweight.
    2. Shave the right flank to expose the skin.
    3. Using a pair of forceps, lift the skin to separate it from the underlying muscle and inject 0.1 mL of the cell suspension (1 x 106 cells) subcutaneously with a 24 G needle. While removing the needle, pinch the injection site for 5 to 10 s so that the tumor cells do not leak out of the injection site.
    4. Revive the mouse with a subcutaneous injection of atipamezole (1 mg/kg) at 0.1 mL per 10 g of bodyweight.
  3. Monitor tumor growth every two days by measuring tumor diameter using calipers. The tumor volume is calculated using the formula v = (ab2)/2, where "a" is the longest dimension and "b" is the perpendicular width, both in mm.
  4. When the tumor volume is at least 50 mm3 (approximately 7 days), euthanize the mouse with CO2. Excise the tumor mass with a pair of sterile forceps and scissors under aseptic conditions and place in DMEM.
  5. In a 6-well culture plate, mince the tumor into small pieces using sterile scalpels. Use a 1 mL syringe plunger as a "pestle" to further disaggregate the tumor.
  6. Add 3 mL of complete DMEM and maintain the cells in an incubator at 37 °C and 5% CO2.
  7. Once the cells adhere to the plate (between one and two days), remove the media, debris, and non-adherent cells by aspirating gently with a sterile Pasteur pipette. Wash twice with DMEM. Take care to not disturb the cells.
  8. Add 1 mL of DMEM and harvest the cells by scraping them with a cell scraper. To select and expand single colonies, count the cells using a hemocytometer and plate 1 cell per well in a 96-well culture plate. Culture cells in DMEM with 200 μg/mL Hygromycin B at 37 °C and 5% CO2.
  9. Change the media and antibiotic every 4 - 5 days. Monitor the cells until they are at about 80-90% confluent. Re-plate the cells on a 24-well culture plate by pipetting to dislodge cells from the well.
  10. Once the cells in the 24-well culture plate are confluent, dislodge them by scraping with a cell scraper and plate them in a 6-well culture plate.
  11. Screen the different clones for PSA secretion, as described in step 1. Cryo-preserve the clone with the highest PSA secretion and use it for future mouse experiments.

4. Implanting the Tumor

NOTE: Each mouse is implanted with 1 x 105 MB49-PSA cells in 50 µL of DMEM blank media in the bladder. Due to the dead space in the catheter, always prepare extra volume (at least 100 µL extra per mouse). An alternative approach would be to use an air filled syringe as described by Kasman et al.5 rather than a filled syringe.

  1. Cells.
    1. One day before implantation, when the cells are approximately 80% confluent, passage the MB49-PSA tumor cells in complete DMEM media (supplemented with 10% FBS, 2 mmol/L L-glutamine, 0.05 mg/mL Penicillin-Streptomycin, and 200 µg/mL Hygromycin B) at 37 °C and 5% CO2 at a split ratio of 1:2.
    2. On the day of the procedure, harvest the cells by scraping them gently with a sterile cell scraper. Mix equal volumes of the cell suspension and a 0.4% trypan blue solution. Count the live cells using a hemocytometer. Do not implant if more than 20% of the cells are dead.
    3. Calculate the amount of live cells required (2 x 106 cells/mL) and transfer them to a 15 mL centrifuge tube. Centrifuge at 250 x g for 5 min at 4 °C and discard the supernatant. Re-suspend the cell pellet in DMEM blank media. Repeat the wash three times to remove all traces of FBS before implantation.
    4. Keep the cell suspension on ice until the mice are ready for implantation.
  2. Allow the 4- to 6-week-old female C57BL/6J mice to acclimatize for one week before the procedure. All animal work must be performed in a biological safety cabinet to maintain sterile conditions.
    1. Weigh each mouse and inject anesthesia (75 mg/kg ketamine and 1 mg/kg medetomidine) intraperitoneally at 0.1 mL per 10 g of bodyweight. Bodyweights should be between 17 and 22 g. Confirm anesthetization by observing no response after a toe pinch.
    2. For identification, mark the ear using an ear punch.
    3. Inject Hartmann's solution or compound sodium lactate intraperitoneally at 0.1 mL per 10 g of bodyweight every 1 - 2 h for hydration.
    4. Apply sterile ophthalmic ointment to both eyes using a sterile cotton bulb, since the blinking reflex is lost under anesthesia and the eyes dry out. Reapply when necessary.
    5. Lay the mice in supine position on paper towels on top of a heat pack (activated by contact with air) to maintain body temperature and tape the hind legs down.
  3. With a finger, apply gentle pressure to the lower abdominal region and collect urine from the bladder into a 1.5-mL microcentrifuge tube. This sample serves as the basal value of urinary PSA.
  4. Withdraw sterile poly-L-lysine (PLL) into a 1 mL syringe and attach a 24-gauge IV catheter, with the needle stylet removed. Apply lubricant to the tip of the catheter and insert the catheter through the urethra into the bladder using forceps to guide the catheterization. Stop when resistance is felt. NOTE: Researchers should discuss with their veterinary staff about the need for the use of a lubricating gel with anesthetic for intravesical instillations and the use of post–procedure analgesics.
  5. Instill 50 µL of PLL slowly, at a rate of 10 µL every 20 s, to avoid vesico-ureteral reflux18. Leave the catheter in the bladder for 20 min with a stopper to prevent out-flow.
  6. After 20 min, remove the catheter and vacate the bladder of any contents by gently pressing on the lower abdominal region. Using an empty 1 mL syringe, flush any remaining contents out of the catheter.
  7. Mix MB49-PSA cells thoroughly by pipetting and withdraw them into a 1 mL syringe. Attach the catheter and apply lubricant. Instill 50 µL of 1 x 105 cells at a slow rate of 10 µL every 20 s. Replace the stopper on the catheter. Give control mice 50 µL of saline.
  8. After 1 h, remove the catheters and vacate the bladder of any contents. Remove the tape on the hind legs and place the mice on their ventral sides. Revive the mice by subcutaneously injecting the reversal drug (1 mg/kg atipamezole) at 0.1 mL per 10 g of bodyweight.
  9. Monitor the mice until motility is observed. Return the mice to their cages.
  10. Upon completion of a tumor detection protocol (sections 5, 7, or 8), euthanize the mice using CO2. Post-mortem tumor detection is described in section 6.

5. Monitoring Tumor Growth with ELISA

NOTE: Tumor presence and growth is monitored in mice by measuring PSA secretion in urine. Researchers should consult with their veterinary staff on monitoring animal health and well-being post-tumor implantation and humane endpoints.

  1. There are two methods to collect urine from mice.
    1. Collect urine overnight by placing a single mouse in an individual metabolic cage designed to separate urine from fecal material. If the set-up does not have refrigeration, add a protease inhibitor (100 µL) into the urine collection tube to prevent degradation of PSA overnight. However, the number of available metabolic cages limits the utility of this method of urine collection.
    2. Where it is logistically impossible to use metabolic cages (such as in the ABSL2 rooms), collect spot urine by using a finger to exert gentle pressure on the lower abdominal region while the mice are anesthetized as described in step 4.2.1. This is usually done before the therapy is instilled. From our experience, at least 150 µL of urine can be collected 1 h after anesthesia.
  2. Immediately place the collected urine on ice. Hematuria may be visible from the second week after tumor implantation. Highly hemolyzed samples may affect ELISA analysis. Therefore, urine must be placed on ice and processed quickly after collection.
  3. Centrifuge the urine tubes at 6,700 x g for 5 min at 4 °C to remove cells or debris.
  4. To normalize the difference in urine output between the mice, aliquot the urine into new tubes and store them at -30 °C until performing the analyses for PSA using an ELISA kit7 and creatinine using an assay kit7. Even though mice do not produce PSA, there are low levels of non-specific binding detected using the ELISA. For samples to be considered positive, the threshold must be set at values greater than in normal mice + 3 Standard Deviations (SD)15.

6. Detecting Tumor Presence with Real-time PCR

  1. At termination, dissect the abdominal cavity of the mouse and locate the bladder. Excise the bladder and place it into a cryovial. Freeze immediately in liquid nitrogen.
  2. Extract the RNA by homogenizing the tissues in an RNA extraction reagent.
  3. Perform reverse transcription and real-time PCR analysis for PSA, as described above in section 2.

7. Monitoring Tumor Growth with Fluorescence Imaging

  1. Label 1 x 107 MB49-PSA cells with a near-infrared fluorescent dye19.
  2. Re-plate and harvest the labeled cells on days 4, 7, 11, 14, 18, and 21 to check if cells retain viability and fluorescence by flow cytometry20.
  3. Implant the labeled MB49-PSA cells in mice bladders, as described above in section 4.
  4. Monitor the tumor growth using a fluorescence imaging system every two days.
  5. On the day of imaging, weigh and anesthetize the mice using a mixture of the anesthetics ketamine and medetomidine (75 mg/kg and 1 mg/kg, respectively), injected intraperitoneally at 0.1 mL per 10 g of bodyweight.
  6. Shave the abdominal area to expose the skin.
  7. Place mice in the imaging chamber and acquire images of the bladders using the software provided with the imaging system.
  8. Revive the mice by subcutaneously injecting a reversal drug (1 mg/kg atipamezole) at 0.1 mL per 10 g of bodyweight.

8. Monitoring Tumor Growth with High-frequency Ultrasound Imaging

  1. Implant tumors in mice, as described above in section 4.
  2. Every two days, monitor tumor growth using ultrasound imaging.
  3. On the day of imaging, weigh and anesthetize the mice using a mixture of the anesthetics ketamine and medetomidine (75 mg/kg and 1 mg/kg, respectively), injected intraperitoneally at 0.1 mL per 10 g of bodyweight.
  4. Shave the abdominal area to expose the skin.
  5. Instill 100 μL of sterile 0.9% saline into the bladder using a 24-gauge IV catheter. The saline will distend the bladder and improve visibility during the ultrasound imaging.
  6. Place the mouse on the ultrasound platform and apply conductive gel to the lower abdomen.
  7. Lower the handheld probe to the skin and acquire B-mode images of the bladder on the imaging platform21.
  8. Revive the mice by subcutaneously injecting reversal drug (1 mg/kg atipamezole) at 0.1 mL per 10 g of bodyweight.

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Wyniki

PSA secretion from MB49 cells was found to vary with the growth media. MB49-PSA is grown in DMEM media because this results in increased PSA secretion (Figure 1A). In order to determine the sensitivity of the PSA ELISA and real-time PCR, different numbers of MB49-PSA-secreting cells were mixed with MB49 parental cells. PSA ELISA detects a minimum of 1 x 105 PSA-secreting cells/1 x 106 cells (Figure 1B), while real-time PCR analysis d...

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Dyskusje

The most critical steps in the protocol are 1) successfully maintaining the tumorigenicity of the cell line; 2) ensuring measurable PSA secretion before tumor cell implantation in mice; 3) generating a single-cell suspension for implantation so as to reduce variation in tumor size; and 4) instilling cells at a slow rate to prevent vesico-ureteral reflux, resulting in tumor cell implantation in the kidney.

After prolonged passage in vitro, MB49/MB49-PSA cells can lose their tumorigenic...

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Ujawnienia

The authors have nothing to disclose.

Podziękowania

This work was funded by a grant from the National Medical Research Council of Singapore (NMRC/CIRG/1335/2012) awarded to Professor Kesavan Esuvaranathan.

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Materiały

NameCompanyCatalog NumberComments
MB49-PSA cellsN/AN/Aref (Wu QH, 2004)
RPMI 1640 mediaHyCloneSH30027.01 
Dulbecco's Modified Eagle's Medium (DMEM) mediaBiowestL0102
Fetal Bovine Serum (FBS) South AmericanBiowestS1810
Fetal Bovine Serum (FBS) South American, PremiumBiowestS181B
Fetal Bovine Serum (FBS)HyCloneSH30088.03 
L-glutamineBiowestX0550
Penicillin-StreptomycinBiowestL0022
Hygromycin BInvitrogen10687-010
free PSA (Human) ELISA kitAbnovaKA0209
TRIzol Reagent for RNA extraction Ambion15596026
High Capacity cDNA Reverse Transcription Kit with RNase InhibitorApplied Biosystems4374967
TaqMan Universal PCR Master MixApplied Biosystems4304437
TaqMan Gene Expression Assay – Mouse ActbApplied Biosystems4331182Mm00607939_s1
TaqMan Gene Expression Assay – Human KLK3Applied Biosystems4331182Hs00426859_g1
C57BL/6J female miceIn Vivos4 - 6 wk old
Anesthesia (75 mg/kg Ketamine and 1 mg/kg Medetomidine)Local pharmacy
Reversal drug (1 mg/kg Atipamezole)Local pharmacy
Ear punchElectron Microscopy Sciences72893-01
Hartmann's solution or Compound sodium lactateB Braun
Ophthalmic ointment - Duratears sterile ocular lubricant ointmentAlcon
Heat pack - HotHands handwarmersHeatmax Inc
Introcan Certo IV catheterB Braun425130024 G x 3/4″
Aquagel Lubricating jellyLocal pharmacy
Poly-L-lysine solution, 0.01%,SigmaP4707
cOmplete, Mini, EDTA-free Protease Inhibitor CocktailRoche4693159001
Quantichrom Creatinine Assay KitBioAssay SystemsDICT-50 
Fluorescent dye - VivoTrack 680Perkin ElmerNEV12000 
RNAlater-ICE Frozen Tissue Transition SolutionAmbion4427575
NameCompanyCatalog numberComments
Equipment and Software
7500 Realtime PCR SystemApplied Biosystems
7500 Software v2.3Applied Biosystems
Metabolic CageTecniplast Vertical type rack for 12 cages
BD FACSCanto I system BD Biosciences
BD FACSDiva software v7BD Biosciences
IVIS SpectrumCT in vivo imaging system Caliper Life Sciences 
Living Image Software v3.1Caliper Life Sciences 
Vevo 2100 imaging system VisualSonics 

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