REAGENT SET UP
Anesthesia preparation Anesthetize the C57BL/6 mice with 2.0% isoflurane and 0.6 L/min oxygen during induction. Reduce the concentration of isoflurane to approximately 1.5 % during maintenance.
PBS (1X solution) Dilute 50 mL of 10 X PBS without calcium and magnesium in 450 mL of autoclaved distilled water. The prepared 1X PBS can be stored at 4˚C for 6 months. CRITICAL STEP The diluted PBS solution should be prepared under sterile conditions and pH adjusted to 7.4.
Human SIRPα/CD172a antibody Reconstitute the lyophilized human SIRPα/CD172a antibody in 0.2 µm filtered PBS to bring to final concentration of 0.5 mg/mL. Aliquot the reconstituted antibodies in sterile prelabeled 500 µL tubes, label and store aliquots for 6 months at -20 °C
Human VEGF R2/KDR/Flk-1 antibody Reconstitute the lyophilized human VEGF R2/KDR/Flk-1 antibody in sterile PBS to a final concentration of 0.5 mg/mL. Aliquot the reconstituted antibodies into pre-labeled 500 µL tubes. Store the labeled tubes for maximum 6 months at -20 o C.
EQUIPMENT SET UP
Transmission electron microscope The transmission electron micrographs presented in this study was captured using Tecnai, G2, 20T, 200kV, FEI make. This microscope has a resolution of 0.28 nm point resolution @ 200kV, acceleration voltage between 80kV to 200 kV and pressure of 10-7 Torr. Energy dispersive x-ray spectroscopy (EDS) and charge coupled devices (CCD) are two detectors available. For this study CCD detectors were employed. The Tecnai software can be controlled by the mouse and keyboard attached to multilingual windows 2000 operating system. The utility requirements however are 100 – 240 v, 50 – 60 Hz single phase, and water cooling system.
Scanning electron microscope The shape and size distribution studies in this study were carried out using Zeiss, EVO-18 with LaB6 filament. This scanning electron microscope has a resolution of 2nm at 30kV – SE, 4.5 nm at 30 kV-BSD (VP mode), 15 nm at 1kV-SE and 10 nm at 3 kV-SE. The acceleration voltage is of 1 kV to 30 kV. The field view is 6mm at analytical working distance and the pressure range from 10 – 400 Pa. Variable detectors are available including BSD multi segment diode, ETSE- everhart- thornley secondary electron detector. The image frame stored has a maximum pixel of 3072 x 3204. The SmartSEM GUL can be operated by both mouse and keyboard attached to a desktop computer installed with windows ultimate operating system. The utility requirements are 100 – 240 v and 50 or 60 Hz single phase.
Atomic force electron microscope The 2D and 3D topographical studies have been carried out using Nanonics Model: MV200. This microscope has a special resolution of 20 nm, Z direction of 0.3 to 0.4 nm and a scan are of 100 µm x 100 µm x 20 µm. The sensitivity of the piezo driven scanner sample stage with closed loop feedback is less than 0.5 nm in XY and less than 0.05 nm in Z for a minimum scan. The tip diameter is 20 nm which can map the surface morphology by three modes – contact mode, non-contact mode and pseudo/intermittent contact mode. In this study the samples were analysed using the pseudo/intermittent contact mode. The capture image can be stored with a maximum frame size of 1024 x 1024 pixel. The nanonics imaging software can be operated by both the mouse and keyboard attached to a Windows 7 professional operating system.
CAUTION AFM experimentation requires an acoustic enclosure with a passive vibration free environment, preferably on an optical bench.
Dynamic light scatter measurement The size and charge characterization was carried out by dynamic light scattering on Malvern Zetasizer Nano ZS. Dilute each of the samples to be analyzed in MilliQ water and carry out the measurements in triplicates at 25 °C.
Confocal microscopy In this study, the physical characteristics and the distribution of the two monoclonal antibodies have been carried out on a Leica TCS SPE confocal scanning laser microscope. This system is equipped with a Leica DMI 4000B microscope with the regular microscopic lens set and a scan head with four solid state laser with wavelength of 405, 488, 532 and 635 nm. The image visualization and capture was processed with the LAS software.
For this study the following setting have been used- magnification 63X, pinhole airy 1.00 A.U, 488nm laser power 13.62% and 33% for SPION and SPION-PEG respectively. GloTrack was imaged with a laser power of 16.77 % for 488nm and 50.25% for 532nm and observed at 63X magnification and pinhole airy 1.00 A.U.
Florescent activated cell sorter The entire florescent activated cell sorting experiment has been carried out on BD FACS Calibur equipped with two lasers of wavelengths 488 nm and 635 nm. This machine has four high performance photomultipliers having bandpass filters including 530 nm (FITC), 585 nm (PE/PI), 670 nm (PerCP) and 661 nm (APC) for absorption. The FACS machine is attached to a BD FACS Station Mac Pro computer for carrying out data analysis and representation. The data analysis was carried out using Cell Quest software.
CRITICAL The recommended single cell suspension should be within the range of 105 to 2 x107 cells/mL. The machine should be placed in a temperature (16 – 29 ̊C) and humidity (10% - 90%) controlled room.
Anesthesia system Connect the oxygen and the isoflurane reservoir to the main induction system (SurgiVet, Highland Medical Equipment). Connect the induction chamber, nose mask and ventilator to the main system using the appropriate tubing, and deliver 1.5 – 2.0% isoflurane with 0.6 L/min oxygen.
MRI Magnetic resonance imaging was carried out using Bruker BioSpec 7.0 tesla 30cm horizontal clear bore magnet small animal imaging system with Paravision 5.1 software (Bruker). This machine is equipped with 7.0 T magnet, radiofrequency resonator signal pre-amplifiers, 35 mm quadrature volume coils for signal transmission, mouse cradle with 35 mm coils, anesthesia unit with triple gas flow meters and isoflurane vaporizer, triple gradient amplifiers, water cooling unit and temperature controllers. Dedicated LINUX run workstations are maintained for monitoring the physiological parameters, data acquisition and processing. The data was acquired using the 1-Tripilot-i.g (scout scan) protocol and fast imaging with steady state precession (FISP) protocol. The scan settings for 1-Tripilot-i.g (scout scan) protocol are, repetition time (TR) of 8.8 msec, echo time (TE) of 1.2 msec, 151 Gaussian pulse of 0.7 msec, 80 mm field view with a total scan time of 26 sec. For the FISP protocol, a gradient – echo sequence with flow compensation was employed and the scan settings are as follows, TE 2 msec, TR 4 msec, field view 80 x 30 mm, matrix size 256 x 256 for a total scan time of 4 min 17 secs.
Preparation of GloTrack for cardiac precursor cell sorting TIMING 19h
1 For mini GloTrack preparation, under sterile conditions transfer 10 µL of commercially available iron oxide (II, II) magnetic nanoparticle in to a 500 µL sterile centrifuge tube.
CRITICAL STEP Vortex the SPION containing bottle briefly prior to aspirating the desired volume of solution.
2 Centrifuge the aliquot of SPION suspension at 8000 rpm for 15 min at room temperature (RT).
3 Under sterile conditions, carefully remove the supernatant and wash the SPION pellet with 100 µL of 70 % ethanol.
CRITICAL STEP Do not disturb the SPION pellet while aspirating the supernatant.
4 Re-centrifuge the SPION-ethanol suspension at 8000 rpm for 15 min at RT.
5 Aspirate out the supernatant under sterile conditions.
CRITICAL STEP Do not disturb the SPION pellet while aspirating the supernatant.
6 Open the flap of the centrifuge tubes in the laminar air flow to allow excess alcohol to evaporate from the tubes. 2 – 3 min.
CAUTION Do not leave the tubes open for more than 2 – 3 min to avoid excess sample drying.
7 Upon drying functionalize the particles using 100 µL of PEG 300 (10 mg/mL). Close, label and seal the tubes tight with parafilm. Allow the tubes to spin in a magnetically stirred water bath at room temperature (RT) for 16 h.
CRITICAL STEP Ensure continuous spinning of the tubes at RT.
8 To the functionalized SPIONs suspension, add 5µg/mL mouse anti human monoclonal SIRPA/ KDR unconjugated antibodies. Incubate the antibodies with the PEGylated SPION at 4⁰C on a magnetically stirred water bath containing ice cold water for 2 h to enhance binding.
CRITICAL STEP Ensure the temperature of the water is maintained throughout the 2 h period.
9 Store the engineered GloTrack at -20⁰C.
PAUSE POINT Prepared GloTrack particles can be stored at -20 ⁰C until use.
Preparation of GloTrack for characterization using confocal microscopy and flow cytometry TIMING 19 h
10 Prepare the GloTrack for confocal imaging and flow cytometry by functionalizing the SPION particles with PEG (10 mg/mL) as mentioned from step1 - 7.
11 To the functionalized SPION particles, add 5 µg/mL mouse anti human monoclonal SIRPA conjugated with PE and mouse anti human KDR unconjugated antibody and incubate for 2 hours on a magnetically stirred water bath containing ice cold water to enhance binding.
CRITICAL STEP It is important to foil wrap the tubes to avoid photo bleaching of the fluorescence dyes conjugated to the antibodies.
12 Allow the antibodies to bind to the open moieties of PEG at 4⁰C as mentioned in step 8.
CRITICAL STEP Ensure the water is maintained at 4⁰C and level is appropriate for continuous spinning of the tubes.
13 After 2 h of incubation add secondary goat anti mouse FITC tagged antibody IgG at 1: 100 dilution to the GloTrack and incubate further for 1/2 h at 4˚C in a magnetically stirred water bath containing ice cold water to enhance binding.
CRITICAL STEP Ensure the water is maintained at 4˚C and level is appropriate for continuous spinning of the tubes in magnetic stirrer.
14 Store the conjugate GloTrack at 4˚C until the samples are analyzed using confocal microscopy or using flow cytometry for 1 – 2 days.
PAUSE POINT Prepared GloTrack particles can be stored at -20˚C for longer storage.
Characterisation of the GloTrack
15 Characterize the prepared GloTrack using the TEM, SEM, AFM, Zetasizer, Confocal or FACS
(A) Morphological characterization of the GloTrack using transmission electron microscopy TIMING 19 h
(i) Transfer SPIONs, PEGylated SPION and the GloTrack into a clean fresh pre-labeled test tube.
(ii) Place these pre-labeled test tubes into a beaker containing methanol of LR grade.
(iii) Fix the test tubes in place with the use of cork and adhesive tapes.
CRITICAL POINT Ensure proper fixation of tubes to avoid cracks and leakages.
(iv) With the samples and tubes in place, transfer the beaker into an ultrasonic bath and sonicate for 15 minutes.
(v) Remove the test tubes carefully and transfer the contents onto pre-labeled clean Petri dishes.
(vi) Using fine tip tweezers lift a 3mm mesh carbon coated copper grid and place it on the sample for lifting it by adsorption.
CRITICAL POINT Carefully place the carbon coated side of the grid by placing it on its side first and then gently lay it over the sample to ensure complete lifting of the sample on to the grid.
(vii) Transfer the sample adsorbed grid on to a lint free paper and place it for a minute in front of infra red light for drying.
CAUTION POINT Avoid excessive dryness as it will lead to artifact formation.
CRITICAL STEP Reverse the sides of the grid every 15 sec for uniform drying.
PAUSE POINT Infra red dried samples can be stored overnight in a vacuum desiccators for examination under electron microscope on the following day.
(viii) Examine the infra red dried samples using Tecnai 20 electron microscope without additional staining.
(ix) Mount the sample loaded carbon coated copper grid on to either a single or double tilt holder.
CRITICAL STEP Use appropriate tweezers for loading samples and handle the grid with care to avoid flexing.
(x) Place the grid loaded holder into the stage of the microscope, as specified in the user operation manual of the microscope.
(xi) Turn on the filament and using the Tecnai user interface open the column.
(xii) Adjust the translating track ball to bring the specimen inside the range of the image on the phosphorous screen and then adjust the eucentric height.
(xiii) Focus the image on the screen.
CRITICAL STEP Use well spread electron beams to avoid any damage to the samples.
(xiv) Record an image of the area of interest using a CCD camera.
(xv) Label the images appropriately with the micron bars, kV and date using the digital software.
CRITICAL Provide the information to the system about the magnification to have the correct micron marker for more accurate measurement of size.
(xvi) After completion of the image capture reset the holder position and adjust the magnification to 4400X, centre and spread the beam to full screen.
(xvii) Close the column valve and turn off the filament.
(xviii) Remove the sample holder from the microscope.
(B) Size estimation of GloTrack using high resolution scanning electron microscopy TIMING 19 h
(i) Prepare samples for SEM as mentioned in Step 15 A i – vii.
PAUSE POINT Infra red samples can be stored over night in vacuum desiccators prior to examination under scanning electron microscope.
(ii) Place the sample loaded carbon coated grid on the ion beam thinning (IBT) holder.
CRITICAL STEP Place and secure the grid in the 3mm slot tightly to avoid drifting during imaging.
(iii) Vent the microscope.
(iv) Carefully load and mount the specimen in the microscope
CAUTION POINT Clean the microscope stage and place a fresh carbon tape for placing the samples to prevent charging and drifting with imaging.
(v) Pump the microscope to obtain the working vacuum level (~10-5 torr).
(vi) Initialize the stage by setting the x, y and z axis to zero.
(vii) Turn on the extra high tension and set it between 5 – 10 kV.
CAUTION POINT High kV should be avoided (more than 10kV) as it can lead to the potential charging and burning effect to the sample while imaging, due to non-conducting surface nature of the sample.
(viii) Choose the secondary image mode and image at low magnification.
(ix) Locate the region of interest by focusing on the specimen surface.
(x) Optimize the imaging parameters such as working distance, filament current, aperture size, probe, focus and also the brightness and contrast of each sample.
(xi) Capture images in different areas of interest.
CRITICAL POINT Capture images on high resolution and on slow scan speed to obtain good resolution images.
(xii) Turn off the extra high tension.
(xiii) Vent the microscope prior to removing the samples
(iv) Pump the vacuum back again.
CRITICAL POINT Handle the samples and the grid with care to avoid damages.
(C) Topographical characterization of the GloTrack using atomic force microscopy TIMING 19 h
(i) Cut the glass slide to the shape to a 1mm x 1mm square using isomet on slow speed.
(ii) Clean the slide on either side with acetone in an ultrasonic cleaning machine for 15 mins.
(iii) Fix the Teflon glue disc on to the glass slide.
CAUTION STEP Ensure a uniform spread of the glue material on the glass slide and absence of air bubbles.
(iv) Take an aliquot of the samples – SPION, PEGylated SPION and the GloTrack in pre-labeled test tube.
(v) Place the pre-labeled tubes in a beaker containing methanol.
CAUTION POINT Fix the test tubes in place with the use of cork and cello tape to prevent any damage.
(vi) Place the beaker then in an ultrasonic cleaner and sonicate for 15 minutes.
(vii) Remove the test tubes from the sonicator and transfer a drop of the solution on to the Teflon glue disk placed on the glass slide.
CAUTION POINT Ensure uniform spread with absence of air bubbles in the solution on the Teflon glue disk on the glass slides.
(viii) Place the samples in front of the infra red lamp for a minute followed by complete air drying in clean atmosphere.
CAUTION POINT Prolonged exposure of the sample to infra red light should be avoided to prevent artifact formation.
PAUSE POINT Samples can be stored overnight in a vacuum desiccator prior to imaging using atomic force microscope.
(ix) Turn on the atomic force microscope, optical microscope, computer on the anti-vibration table.
(x) Open the interface and the desired Nanonics (v NWS11_1.0¬_03_07_14) software for acquisition.
(xi) Place the samples on the vibration free pizeo stage.
(xii) Choose the intermittent AFM mode.
(xiii) Mount the tip on the upper scanner of the microscope.
(xiv) Tune the frequency of the cantilever of the AFM scanner by matching it with the frequency of the tunning fork as supplied by the manufacturers.
(xv) Locate the set points.
(xvi) Check the read error channel, which should preferably be 0.3.
(xvii) Use the optical light microscope to determine the region of interest.
(xviii) Then focus on the tip of the region of interest using the X and Y movement of the stage.
(xix) Approach the AFM tip on the sample using the coarse approach with high speed (900Hz – 1250Hz) till the distance between the samples is 1 – 2 mm or alternatively with fine approach of 200 – 300 Hz under slow speed.
CRITICAL POINT Ensure the engagement of the probe on the surface by both the hardware and software. On the scanning probe microscope (SPM) controller, check for the illumination of the feedback (FB) and on the software check for the fluctuation of the error signal needle around zero.
(xx) Prior to beginning the scan, pre-set all the scan parameters including area, pixel size, dwell time, scan direction, location of scanner and also the scanner mode.
(xxi) Begin scanning the area of interest.
CRITICAL POINT Turn off the optical light to minimize the sample drift.
(xxii) Check the image quality and save the file.
PAUSEPOINT Captured images can be saved for analysis at a later stage.
(xxiii) When desired convert the file to the required format for 2D and 3D surface image profiles.
(xxiv) Using the Nanonics software (v NWS11_1.0¬_03_07_14) to analyze the results for roughness, 2D and 3D surface topography etc.
(D) Surface charge and size estimation of GloTrack using Zeta sizer
TIMING 1 h
(i) Transfer 20 µL of SPION, 500 µL of SPION PEG and 500 µL of GloTrack prepared with unconjugated antibodies into a clean and pre-labeled 1.5 mL centrifuge tube.
(ii) Make up the volumes in each of these tubes to 1 mL by diluting the samples with water. Gently vortex the samples prior to analysis.
(iii) Load the samples on to a 1mL disposable syringe.
CRITICAL STEP Avoid air bubble formation. Gently tap to release if any.
(iii) Prior to measuring the samples, clean inside of the zeta cell thoroughly by flushing it with water, followed by ethanol and finally with water.
(iv) For zeta size measurement, transfer the samples into a cuvette and fill between 10 – 15 mm in height.
(v) Gently tap to remove any presence of air bubble in the cuvette.
(vi) Place the cuvette in its slot in the machine.
(vii) Run the samples after loading the desired protocol that is preset in the machine.
(viii) Record the sample size values, histogram from the generated report following measurement.
(ix) For Zeta potential estimation, use the previously prepared sample and load it into the zeta cuvette.
(x) Ensure the sample is filled completely in the cuvette and submerging the electrodes.
CRITICAL POINT The sample levels on either side of the Zeta cell should be equal and sealed.
(xi) Clean the sides of the cuvette and place it in its slot.
(xii) Run the samples in triplicates with a zeta protocol.
(xiii) Generate histogram with the acquired sample values from the measurement file summary.
(E) Distributional analysis of monoclonal SIRPA/KDR antibodies on GloTrack using confocal microscopy TIMING 3 h
(i) Take an aliquot of 50 µL of SPION in a clean 500 µL tube. Vortex the SPION suspension at high speed for 10 seconds. Place an aliquot of 10 µL on a clean glass slide; allow it to partially air dry for 1 -2 min and then place a cover slip over it.
CRITICAL STEP Allow partial air drying to avoid complete spreading of the particles around the cover slip. Fix the cover slip and seal to avoid it from being disturbed while acquiring.
(ii) Similarly, take an aliquot of PEG coated SPION in a clean 500 µL foil wrapped tube to which add goat anti mouse FITC in the ratio of 1:100. Allow the labeled antibody to non-specifically bind to the open moieties of the PEG coating for 30 min at 4⁰C. Remove 10 µL of the labeled functionalized SPION and place it on a clean pre-labeled slide.
(iii) Allow it to partially air dry, place a cover slip and seal the edges.
CRITICAL STEP Do not allow the SPION – PEG mixture to excessively dry.
PAUSE POINT The slides can be foiled wrapped and stored at 4⁰C for a maximum of 2 days prior to imaging under confocal.
(iv) Prior to analyzing the florescent labeled GloTrack, bring the tubes to room temperature. Place an aliquot of the fluorescent labeled GloTrack mix on the glass slide in a dark room. Allow the GloTrack mixture to partially air dry and then place the glass cover slip over the aliquot.
CRITICAL STEP Do not allow the GloTrack mixture to excessively dry. Seal the edges and wait prior to observation.
PAUSE POINT The slides can be foiled wrapped and stored at 4⁰C for a maximum of 2 days prior to imaging under confocal.
(v) Analyze the SPION particles under transmission light, while the FITC labeled SPION-PEG using 488 laser and transmission light and the GloTrack using 488 and 544 laser along with transmission light. Capture the images using sequential scan with the selected lasers. PAUSE POINT The post analysis can be carried out later by the investigator.
(F) Monoclonal SIRPA/KDR antibody evaluation on the GloTrack using flow cytometry TIMING 2 h
(i) Transfer two aliquots of 50 µL of SPION each in to two 500 µL centrifuge tube and vortex it at high speed. To one of the aliquot of SPION add 5µL of FITC labeled goat anti mouse IgG and incubate in dark for 30 min.
CRITICAL STEP Label the tubes appropriately and foil wrap the tube with the fluorescent dye.
(ii) Transfer two aliquots of SPION PEG each 250 µL into clean centrifuge tubes and vortex gently. To one aliquot of the functionalized SPION add goat anti mouse IgG FITC secondary antibody and incubate in the dark for 30 min.
CRITICAL STEP Label tube appropriately and foil wrap the tube with the fluorescent dye.
(iii) Take 250 µL of GloTrack prepared as explained in step 10 – 14.
CRITICAL STEP Foil wrap the tube to avoid photobleaching and label accordingly.
(iv) To another clean centrifuge tube transfer 250 µL of GloTrack prepared as explained in steps 1-9.
(v) After 30 mins of incubation in dark, transfer all the aliquots of SPION, SPION PEG and GloTrack into pre-labeled FACS tubes and make up the volume to 500 µL with PBS.
CRITICAL STEP This step should be carried out in the dark and minimum exposure to light is essential. Each of these individual tubes should be labeled with their corresponding sample names and should be treated as test groups. Vortex each of these samples prior to acquiring.
(vi) Similarly, transfer all the aliquots of SPION, SPION PEG and GloTrack into fresh, clean FACS tubes and make up the volume of each to 500 µL with PBS.
CRITICAL STEP Label these individual tubes with the corresponding sample name and retain each of these samples as controls.
(iii) Set the FACS machine as per the manufacturer’s instructions and carry out the analysis using the Cell Quest software.
(iv) Acquire the control unconjugated SPION, SPION PEG and GloTrack samples initially at low voltage and adjust the peaks for the individual dyes – FITC and PE by altering the voltages for aligning the positive and negative region.
CRITICAL STEP Care should be taken while adjusting the voltages.
(v) Set the protocol upon adjusting the voltages.
(vi) Run the remaining conjugated SPION, SPION PEG and GloTrack.
(vii) Save the data of each samples.
PAUSE POINT Complete the data analysis at the investigators convenience.
(viii) The analyzed data can be represented as histogram or contour plots.
(G) MRI of the biodistribution of GloTrack Nanoparticles in C57BL6 mice TIMING 7 h
(i) Anesthetize C57BL6 mice with 1.5 – 2 % isoflurane and 0.6 L/min oxygen in the induction chamber.
CRITICAL STEP Complete sedation is crucial; observe for relaxed muscles and absence of pedal, palpebral and corneal refluxes.
(ii) Use one french catheter (26 gauge catheter) and a 23 gauge needle for venipuncture prior to insertion of a tail vein catheter into the anesthetized mice.
CAUTION Use a mouse tail illuminator (MIL) to highlight the tail veins for insertion of the catheter.
CRITICAL STEP Ensure the catheter is in place.
(iii) Tape the mouse tail in place using HY-TAPE of 3 – 4 mm width around the catheter and tail to prevent slipping of the catheter.
(iv) Restrain the anesthetized mouse and place it within the MR probe. Maintain the temperature of the imaging scanner at at 37 ºC and position the animal in supine position with the head first.
(v) Pre-scan the animals prior to the administration of the GloTrack to obtain base line values.
(vi) Following base line readings, resuspend the GloTrack in saline to bring it to a final concentration of 1 mg/Kg body weight of each animal.
(vi) Using a 1mL syringe administer 100 µL of the final resuspended GloTrack through the tail vein.
(vii) Take a series of images over a 90 min period to determine kinetic distribution of the GloTrack to different tissue parts.
(viii) Carry out the post processing of the images using Paravision (v5.1) software (Bruker Biospin).
CRITICAL STEP Generate a differential image (post contrast at desired time minus pre-contrast image data set for the same slice) to determine the migration and entrapment of the GloTrack in different locations assessed based on the decrease of the MR signal intensity. A pre-contrast morphological image can be used for anatomical orientation.
(ix) Statistically analyse the data using ANOVA for migration kinetics, and a Student’s t-test for MRI signal intensities in measured tissues (e.g. heart, kidneys, lung, liver, muscle).