Dye-Trak® is a registered trademark of Triton Technology, Inc. US Patents 5,230,343 and 5,253,649 other US and Worldwide Patents Pending. Copyright 1991, 1992, 1993, 1994 by Triton Technology, Inc.
This guide provides an overview of the utilization of Dye-Trak® microspheres for measurement of regional blood flow in experimental animals. Dye-Trak® microspheres offer a non-radioactive alternative to the traditional radio-isotope labeling of microspheres. While every investigator will design his or her own experimental protocol, certain steps in the injection, retrieval and quantification of these spheres will be shared by all the Dye-Trak® microsphere users. This guide is principally intended for the laboratory staff responsible for setting up and executing the experimental procedures. The scientific basis for use of this product has been established in the literature (1).
We welcome your suggestions and comments regarding this guide. Please address questions which are not discussed herein via FAX at (858)272-1451 or e-mail: triton2@cts.com. We intend to revise this document periodically and add useful information which Dye-Trak® microspheres users may contribute.
The first step towards the successful use of Dye-Trak® microspheres is to design a written protocol for the specific experiment to be conducted. The protocol should identify the equipment and supplies needed to carry out the planned work. We recommend following the steps described in section 4.1 before you commence any in vivo application of the Dye-Trak® microspheres. This section provides information on the equipment and supplies involved in the use of Dye-Trak® microspheres. Specific manufacturers, model numbers and vendors of this equipment are provided for your convenience. While we have been careful in their selection, their inclusion does not imply any warranty by Triton Technology, Inc. regarding their safety, use or quality.
2.1 REQUIRED EQUIPMENT & SUPPLIES
This section lists specific manufacturers which have been used in processing Dye-Trak® microspheres. It should be understood that equivalent instruments and equipment may be substituted for any of the listed items on the following page.
Equipment:
UV/VIS Spectrophotometer (should be capable of generating ASCII data files to computer or floppy disk)
100uL to 200ul UV/VIS spectrophotometer microcuvette for spectrophotometer
Ultrasonic Processor with Probe Tip, 70-Watt (COLE-PARMER P-04714-00 or equiv.)
Includes 6mm Titanium tip
3 mm Titanium Ultrasound Probe Tip (250ul - 10ml sample volume)
(COLE-PARMER P-04712-12 or equiv.)
Ultrasonic Processor Footswitch (optional, recommended) (COLE-PARMER P-04712-05 or equiv.)
Ring-Stand with clamps, bars, etc.
200ul Adjustable Pipetter with disposable tips
Bench-Top Centrifuge, with buckets and holders compatible with 50ml and 15ml centrifuge tubes.
Vacuum Aspirator (with waste collection reservoir)
Pasteur pipettes with bulbs
Becton Dickinson Cell Culture Insert, 3.0 m m or 8.0 m m PET membrane, catalog # 3096 or # 3097
50ml Conical Polypropylene Screw-top Centrifuge Tubes (FALCON 35-2098 or equiv.)
15ml Conical Polypropylene Screw-top Centrifuge Tubes (FALCON 35-2096 or equiv.)
(Note: DO NOT use polystyrene centrifuge tubes, this plastic is too brittle!)
Tissue & Blood Processing Equipment, Chemicals and Reagents:
Reagents:
All reagents should be A.C.S. grade or better.Denatured Ethyl Alcohol, 1 liter
N,N-Dimethylformide, spectrographic grade, 1 liter
Tweenâ 80, 25ml
Tritonâ X-100, 1 Liter
Potassium Hydroxide, pellets, FW 56.11, 1kg
Hydrochloric Acid, 37%, 500ml
Sodium Azide, FW 65.01, 5 gram
Thimerosal, FW 404.8, 1 gram
Sodium Chloride, FW 58.44, 100 gram
Processing Reagents (store at room temperature):
Blood Hemolysis Reagent:
Add 1 liter of 10% Triton X-100 solution (see below) to a 2 liter glass beaker. Place the beaker on a magnetic-stirring plate and begin stirring with a magnetic stirring bar. Add 200ml of Ethanol to the solution with stirring. Store the solution in plastic bottles identified as "Blood Hemolysis Reagent (BHR)".
Alkaline Digestion Reagent (1.0 M KOH):
Add 2000ml of distilled water to a 2-liter glass beaker. Place the beaker on a magnetic-stirring hot plate. Place a magnetic stirring bar into the water and begin stirring at a fairly rapid rate. Add 112.22 gram of Potassium Hydroxide pellets to the water and stir until the solution becomes clear. Turn off the heat and cool to room temperature with continuous stirring. Store the solution in plastic bottles identified as "Alkaline Digestion Reagent (ADR)". Caution: This is a very caustic solution, handle with care !
Acidified Ethanol Reagent:
Prepare a stock solution of acidified ethanol by adding hydrochloric acid (HCl, 37%) to a beaker of ethanol, 0.2% (volume/ volume). For example, add 2ml of HCL to 1000ml of ethanol. Store the solution in a 1 liter plastic bottle identified as "Acidified Ethanol (AE)"
10% Triton X-100 Reagent:
Add 1800ml of distilled water to a 2-liter glass beaker. Place the beaker on a magnetic-stirring hot plate and heat the water to approximately 50° C. Place a magnetic stirring bar into the water and begin stirring at a fairly rapid rate. Add 0.20 gram Sodium Azide to the distilled water. Add 200ml of Triton X-100 to the hot water and stir the viscous Triton X-100 into water until the solution becomes clear. Turn off the heat and cool to room temperature with continuous stirring. Store the solution in plastic bottles identified as "10% Triton X-100".
15% Triton X-100 Reagent:
Add 1700ml of distilled water to a 2-liter glass beaker. Place the beaker on a magnetic-stirring hot plate and heat the water to approximately 50° C. Place a magnetic stirring bar into the water and begin stirring at a fairly rapid rate. Add 0.20 gram Sodium Azide to the distilled water. Add 300ml of Triton X-100 to the hot water and stir the viscous Triton X-100 in water until the solution becomes clear. Turn off the heat and cool to room temperature with continuous stirring. Store the solution in plastic bottles identified as "15% Triton X-100".
10% Tweenâ 80 Solution:
Add 90ml of distilled water to a 1-liter glass beaker. Place the beaker on a magnetic-stirring hot plate and heat the water to approximately 50° C. Place a magnetic stirring bar into the water and begin stirring at a fairly rapid rate. Add 0.05 gram Thimerosal to the distilled water. Add 10ml of Tweenâ 80 to the hot water and stir the viscous Tweenâ 80 in water until the solution becomes clear. Turn off the heat and cool to room temperature with continuous stirring. Store the solution in plastic bottles identified as "10% Tweenâ 80". Note: The shelf life of this reagent is approximately 2 weeks.
0.05% Tweenâ 80/Saline Solution (Microsphere Carrier Solution)
Add 0.50ml 10% Tweenâ 80 solution to 99.5ml sterile saline solution and mix by stirring. Make the reagent as needed for use as an injectate solution. Store in plastic bottles identified as "Saline plus 0.05%Tweenâ 80 (MCS)".
* Indicates US part numbers for 120 VAC operation.
2.2 REAGENTS
You will need to prepare some stock solutions of commonly available reagents for use in this procedure, including:
A sufficient supply of Dye-Trak® microspheres should be maintained to execute all planned experiments. Unlike radioactive microspheres, Dye-Trak® microspheres do not degrade over short periods of time. You may, therefore, take advantage of quantity discounts and stock enough spheres for several months.
The amount of microspheres required per experiment is determined by the following rules. The dose must be such that the smallest sample of tissue which will be quantified receives no less than 400 spheres in order to meet established statistical requirements. Moreover, in the case of violet microspheres, a minimum of 750 microspheres are needed to exceed the typical noise figure of 0.05 AUs in the spectrophotometer.
The maximum numbers of microspheres which can be processed in 100 ul of DMF without exceeding the 1.3 AU linearity limit, are approximately:
White: 6,000
Yellow: 3,000
Red: 6,000
Blue: 12,000
Violet: 15,000
The concentration of microspheres in suspension may easily be changed. If a lower concentration is required, simply dilute a portion of the suspension in an appropriate volume of saline. If a higher concentration is desired, the contents of the bottle should be transferred and centrifuged in a conical tube at 2,000 g. The supernatant fluid to be removed is pipeted out to achieve the desired concentration. Once again the spheres suspension should be vortexed and sonicated prior to injection.
The primary applications of Dye-Trak® microspheres are local organ regional blood flow distribution, whole body blood flow distribution and cardiac output determination. Blood flow distribution within an organ may be determined by local injection of microspheres into the vessel perfusing the organ. Whole body distribution and cardiac output may be determined by central injection of microspheres into the left atrium along with a calibration reference blood sample withdrawal.
Animals used shall be handled in accordance with the guidelines of the animal welfare regulations of the American Physiological Society or prevailing rules in the state, country or institution of use.
Record the part number and lot number of each product for every experiment. This will allow you to trace the quality assurance statistics maintained for every batch of microspheres.
A local injection of Dye-Trak® microspheres into a vessel perfusing a specific organ will allow determination of relative regional blood flow distribution within the organ. By measuring total organ inflow with an independent technique (i.e. flowmeter), absolute regional blood flow may be determined. This technique can be used with a local catheter injection in the perfusing vessel or in a cannulated externally perfused vessel.
Select the amount of microspheres required proportional to the perfused organ mass as described in Section 2.3. The following example describes the procedure for left anterior descending (LAD) coronary artery injection in a 20 kg dog.
The number of injected Dye-Trak® microspheres varies between Red, White, Violet, Yellow and Blue to account for their different absorbance characteristics. On the day of the experiment, the previously portioned microspheres may be further diluted with saline (containing 0.02% Tween 80) or concentrated to a total volume which will not affect the subject's hemodynamics. Ultrasonication for 1 minute immediately prior to injection is recommended. Each microsphere injection is followed by a flush of saline solution.
It may be desirable to delineate the perfused bed prior to euthanasia. For example, methylene blue may be injected into the LAD line. The entire delineated area may then be removed and further processed.
Microspheres are injected through a teflon catheter into the left atrium. A second teflon catheter is inserted into the descending aorta for withdrawal of reference blood samples.
For each measurement in a 20 kg animal, approximately 1.8x 106 (yellow, white) to 7x106 (Violet) Dye-Trak® microspheres are injected. On the day of the experiment, the previously portioned microspheres may be further diluted with saline (containing 0.02% Tween 80) or concentrated to achieve a volume compatible with the animal's size, and ultrasonicated for 1 min immediately prior to injection. Each microsphere injection is followed by a flush of saline solution.
Reference arterial blood samples are collected through the aortic catheter starting 10 s before injection of the microspheres and continuing for 100 s at a rate of about 6 ml/min for a 15 kg animal.
There are inherent differences in the dye-release microspheres which should be noted, particularly by users of radio-labelled tracers.
Certain dyes used for demarcating ischemic and necrotic tissue adhere to polystyrene microspheres. This can cause substantial artifacts in the sample's absorption spectra. In certain cases, such as with Evans Blue, the bonding is weak and the extraneous dye is washed away during the rinsing of the microspheres in ethanol (Section 4.3 and 4.4). Other dyes, such as triphenyltetrazolium chloride (TTC-red), are known to bond strongly to the polystyrene microspheres, remaining even after ethanol washing.
Dye-Trak® microspheres should not be frozen prior to administration. Tissue samples containing these microspheres may be frozen. Freezing below 4 deg. C. may cause substantial cracking of the particles and their subsequent loss in the filtration procedure.
Dye-Trak® microspheres will deform above 90C, potentially releasing some of their dye. Elevated temperatures above this limit should, therefore, not be used in conjunction with this product.
This section describes the processing of harvested tissue samples and reference withdrawal blood samples to spectrophotometrically quantify the amount of dye in each of them.
Dye-Trak® microspheres are quantified by their dye-content. The dye is recovered from the microspheres by adding 100 ul DMF as a solvent with a Hamilton syringe (Hamilton). To ensure complete recovery of the dye, Dye-Trak® microspheres and DMF are put together into a conical centrifuge tube and vortex-mixed for 30 s. The tube is then centrifuged (5min, 2000g) and the dye-solution transferred into 4 ml glass tubes (Baxter Scientific). Finally, the transferred dye solution is separated from remaining particles and microspheres by centrifugation (3 min, 2000g) to minimize scatter in the subsequent spectrophotometry.
The completeness of dye removal from the microspheres has been tested by adding increasing amounts of the DMF solvent from 50 to 800 ul in 8 samples of dried Dye-Trak® microspheres, and by exposing the Dye-Trak® microspheres to DMF for increasing durations from 20 s to 1 hour. Confirming the completeness of dye removal from the microspheres by 100 ul DMF for a few minutes (30 s vortex agitation plus 3 min centrifugations), no increase in dye-removal occurred with increasing amounts of DMF and increasing duration of exposure to DMF.
The photometric absorption of each dye-solution is determined by a spectrophotometer (Model 8452A, Hewlett Packard or equivalent, wave length range: 320-820 nm, 2 nm optical band width). In a manner similar to the overlap correction in counting the RM, the composite spectrum of each dye- solution is resolved into the spectra of the single constituents by a matrix inversion technique.(7) The absorption spectrum of each dye is measured separately and served as a reference for the matrix inversion, determining the contribution of each color to the measured composite spectra at five fixed wavelengths: 370, 448, 530, 594 and 672 nm. Within 10 seconds, 100 single measurements of 0.1 s duration are performed and averaged such that the standard deviation of each absorbance value is less than 0.1% of the mean. The amount of dye or the number of Dye-Trak® microspheres in a given sample is adjusted to achieve absorbance values of no more than 1.3 AU (absorbance unit, 1 AU = -1g(10% light transmission/100%)) and thus assure the linearity between absorbance and dye concentration according to the Lambert-Beer law. Samples with absorbances higher than 1.3 AU are diluted and analyzed again. The lower limit of detection which could be reliably distinguished from background noise is 0.05 AU.
This procedure is designed to give first-time microsphere users hands-on experience in successfully handling microspheres, reagents and spectrophotometer prior to initiating animal experiments.
1) Agitate a bottle of Dye-Trak® microspheres by vortex mixing for 30 seconds. Do not over-agitate, as this will cause foam in the vehicle. Surface tension in these bubbles may trap as much as 15% of the suspended microspheres.
2) Using a microsyringe, extract 100 microliters of the agitated suspension. This volume will contain approximately 300,000 spheres.
3) Transfer the contents from the microsyringe into a 15 milliliter test tube.
4) Add 9.9 milliliters of saline with .01% Tween 80 to the test tube. Cap and agitate the suspension. You will now have 10 mililliters of diluted suspension.
5) Extract 100 microliters of the newly diluted suspension using a clean micropipette. This volume will now contain 3,000 spheres.
6) Transfer this volume into a small (i.e. 1.5 ml Eppendorf) centrifuge tube. Centrifuge for 4 minutes at 2000 g's. The microspheres will now lie in a pellet at the bottom of the tube.
7) Carefully pipette the supernatant fluid and discard it.
8) Dry the microspheres.
9) Add 300 microliters of 100% Dimethyl-formamide (DMF). Cap the tube and vortex mix for one minute. This will extract and dissolve the dye.
10) Centrifuge for 4 minutes at 2000 g's. The microspheres will now lie again in a pellet at the bottom of the tube.
11) Carefully pipette the DMF, now bearing the dye in solution into a cuvette for spectrophotometric analysis. Repeat the above procedure for all 5 dyes, using clean glassware each time. After you have successfully obtained individual spectra for each dye, you may combine different microspheres from your diluted stocks obtained in step 4. By repeating steps 5 through 10 with a mixture of spheres, you will obtain a composite spectrum.
4.2 CHANGING MICROSPHERE VEHICLE
Dye-Trak® microspheres are supplied in a concentration of 3 million particles/ml. The vehicle is saline with .01% Tween 80 added as a surfactant which prevents agglomeration and with .01% Thimerosal as a bacteriostat. In the event it may be necessary to change the suspension vehicle, this can be easily accomplished following the steps below. Reasons for changing the vehicle include contamination of the product, need to change concentration of microspheres or undesireable effects of the surfactant and bacteriostat agents supplied.
Note the lot number of the product prior to removing the particles from the original package. Transfer the contents of the bottle into a conical centrifuge tube. Centrifuge for 5 minutes at 2,000 g. Pipette the vehicle out and add 20 ml of absolute ethanol. Vortex mix the conical tube and its contents for 2 minutes. Repeat the centrifuge, wash and agitate steps once more. Pipette the ethanol out and air dry the microspheres for 30 minutes at room temperature. Add the desired vehicle (i.e. Dextran), ultrasonicate and store in a sealed, labelled container.
1.) Harvest tissue samples from the tissue beds of interest. Tissue samples may be stored at 0-4° C for short-term storage or frozen for long-term storage. Alternatively, the tissue samples may be fixed with formalin. Note: If any other tissue-fixing agents are to be used, they should be tested throughout the tissue processing procedure for any negative microsphere/dye interactions. Spectrophotometric analysis must confirm dye absorbance profile and intensity stability before use.
2.) Tissue samples must be processed in new disposable polypropylene centrifuge tubes appropriate to the tissue sample size. Polystyrene centrifuge tubes should not be used as they are quite brittle and tend to crack during multiple centrifugation steps. Tissue samples 3 gram or less should be processed in the smaller 15mL centrifuge tubes and tissue samples from 3-10 gram should be processed in the larger 50mL centrifuge tubes. (See note below for tissues sample-types that do not follow this general rule.)
3a.) Each small tissue sample (3 grams or less) is placed in a pre-weighed ('tared') 15mL polypropylene centrifuge tube. (Weigh the centrifuge tubes with the caps removed). Push or briefly centrifuge each sample to the bottom of its tube. The centrifuge tube is then re-weighed to determine the wet-weight of each tissue sample. Each tube should be labeled with an identification number or name written on the outside surface. The 'wet weight' of each sample should then be recorded along with the Sample Identification information. This information will be used later for blood flow calculations.
3b.) Larger tissue samples (3-10 grams) should be placed in a pre-weighed ('tared') 50mL polypropylene centrifuge tube and pushed or briefly centrifuged to the bottom of the tube, (Weigh the centrifuge tubes with the caps removed). The centrifuge tube is then re-weighed to determine the wet-weight of each tissue sample. Each tube should be labeled with an identification number or name written on the outside surface. The 'wet weight' of each sample should then be recorded along with the Sample Identification information. This information will be used later for blood flow calculations.
4.) The following Internal Control (QC) step is optional, but is strongly recommended as an internal check of 'microsphere recovery'. Add 5,000 microspheres of a color that was not used in the blood flow protocol (preferably Violet 694nm), to each sample processing tube. The quality control spheres are used as a control for any possible fractional loss of microspheres during sample processing. ( This costs less than 3 cents per tissue sample! ) Make up a solution with a concentration of 50,000 control microspheres per mL, using the Microsphere Carrier Solution (MCS) to dilute the factory concentration of 3 million microspheres per mL. Then, use a 100 uL 'repeating-pipette' to add 5,000 of QC spheres to each tissue processing tube prior to hydrolysis.
5a.) Add 6mL of Alkaline Digestion Reagent (ADR) (see Reagent Preparation section) to each <3-gram sample in a 15mL centrifuge tube. Place the screw cap on each tube and tighten snugly. (IMPORTANT: Do not use more than 6mL of ADR in the 15mL tubes.)
5b.) Add 20mL of Alkaline Digestion Reagent (ADR) (see Reagent Preparation section) to each >3 gram sample in a 50mL centrifuge tube. Place a screw cap on each tube and tighten snugly. (IMPORTANT: Do not use more than 20mL of ADR in the 50mL tubes.)
Go to "Overnight Alkaline Hydrolysis at 60° C", Section II of the Procedure and continue using the sample processing instructions. From this point onward the processing steps for tissue and blood is the same.
Blood samples must be processed in new 50 mL disposable polypropylene centrifuge tubes. Polystyrene centrifuge tubes should not be used as they are quite brittle and tend to crack during multiple centrifugation steps. Reference Blood Samples larger than 20 mL should be split and processed in multiple 50 mL centrifuge tubes.
5,000 microspheres of an unused color (preferably Violet 594nm ), is added to each blood sample as an internal microsphere control. (see tissue processing step 4) The control microspheres need to be added to a large blood sample before splitting and evenly distributed throughout the blood by vortex mixing inversion mixing. Split samples will be recombined later when washing with ethanol to recreate a single blood sample.
Add Blood Hemolysis Reagent (BHR) (see Reagent Preparation section) to the top of each tube and mix by inversion. This will be referred to as Quantity Sufficient (QS) in the flow charts. Preferably, this should be done immediately after reference sample collection to prevent blood coagulation. Otherwise, add 10 mg/mL EDTA to each reference blood sample to prevent blood coagulation, with repeated-inversion mixing.
Centrifuge the tubes for 15 min. at 1,500g and aspirate the supernate to a level safely above a not easily visible pellet. If in doubt, aspirate no lower than the 10 mL volume mark.
Add 20mL of Alkaline Digestion Reagent (ADR) (see Reagent Preparation section) to each 'reference-blood' 50mL centrifuge tube. Place a screw cap on each tube and tighten snugly. (IMPORTANT: Do not use more than 20mL of ADR in the 50mL tubes.)
Go to "Overnight Alkaline Hydrolysis at 60° C" Section II of the Procedure and continue using the 50 ml sample tube processing instructions. From this point onward the processing steps for tissue and blood is the same.
Section II : Overnight Alkaline Hydrolysis
Place the tubes in a general-purpose laboratory oven at 60° C and allow the tissue/blood to digest overnight ('alkaline hydrolysis'). After overnight digestion, remove the sample tubes from the oven, briefly loosen the screw caps to vent air pressure, re-tighten the screw caps snugly, then, thoroughly vortex-mix the contents of the tube for approximately 15-30 seconds. The tissue/blood samples in each tube should completely homogenize into suspension, (only small particles of fatty white debris should be visible in the tissue samples). Return the sample to the oven for an additional hour of digestion.
7.) After an additional hour of digestion, repeat the vortex steps described above. Visually inspect the samples again. If undigested pieces of tissue remain visible, continue digestion at 60° C throughout the day with intermittent vortex mixing as required. The break-up of these pieces can be accelerated with a brief 'sonication' (see Note 4). After each tube is 'sonicated', rinse the sonicator probe-tip with distilled water back into the sample tube so that no spheres are lost.
8.) Remove all tubes from the oven and fill each tube by adding 50° C distilled water to bring the solution level to the top of each tube, QS (quantity sufficient). Do not overfill!. Mix the contents by repeated inversion.
9.) Centrifuge the sample tubes for 15 min. at 1,500g (typically 2500 rpm) and aspirate the brown/green supernatate to a level safely above the visible pellet in each tube (0.5-1 cm above the pellet).
Break-up of Tissue Debris with Sonication:
10.) Re-suspend the tissue/blood pellet with the 10% Triton X-100 Reagent warmed to 50° C. Use 12 mL in the 15mL tubes or 40 mL to 50 mL tubes. Don't over-fill the tube as the sonicator tip will cause the liquid to overflow and spheres will be lost! It is important to use the sonicator to thoroughly break-up the microsphere pellet at the bottom of the tube. While sonicating, move the probe-tip repeatedly from the bottom to the top to the bottom of the centrifuge tube, thoroughly sonicating all the material into a homogeneous suspension. Wash sonicator probe-tip back into sample tube with distilled water after each sonication step (using wash bottle).
Note: When working with brain and spinal cord tissues, substitute. 15% Triton X-100 Reagent and perform two cycles of re-suspension, sonication, and centrifugation.
11.) Centrifuge the tubes for 5 min. at 1,500g and again aspirate the supernate in each sample tube to a level safely above each visible pellet.
12.) Repeat Steps 10 and Step 11 two more times, substituting Acidified Ethanol Reagent (AE) as the wash reagent (instead of using Triton-X-100 reagent). After two cycles of re-suspension, sonication, and centrifugation, most tissue samples will have a very small pellet, containing only the colored microspheres and very small amounts of tissue debris.
Section III: Microsphere Recovery
There are two alternate ways to analyze the recovered colored microspheres from this point onward:
Option 1 requires no filtering and is recommended for most users. This procedure relies on the 'evaporative drying of the recovered spheres and is best done at room temperature overnight or in a temperature-controlled oven at 50° C.
Option 2 is recommended for users requiring expedited analysis of colored microsphere samples. The procedure uses small 'tissue-culture' filter inserts to recover the spheres from the clarified digestate solution. This procedure is fast and gives very clean analysis samples, which is particularly important for low blood flow studies. The filters cost approximately $3 each, but they can be reused several times with proper cleaning and handling (see Hints and Notes below)
OPTION 1: Non-Filter (Evaporation) Recovery of Microspheres and Dye
Allow the microsphere sample pellets to evaporate dry overnight at room temperature or ccelerate evaporative drying at no greater than 50° C. Caution: At higher temperatures the plastic centrifuge tube can absorb some of the eluted dye.
Add 200ul of DMF reagent to each tube and vortex-mix. Let each tube stand for 15 min to allow the solvent to elute the colored dye from the microspheres. Vortex-mix the tubes again. Centrifuge the tubes for 5 min at 1,500g to pellet the clear microspheres and tissue debris. The supernate solution will contain the extracted dye for analysis.
Prior to the analysis of the DMF elution samples, perform a baseline spectrophotometric absorbance run with clear DMF across the entire wavelength spectrum of interest.
Analyze each sample by absorbance spectrophotometry using an appropriate volume microcuvette (100uL to 200uL). Use a new 'narrow tip glass transfer pipette' to transfer the sample fluid to the microcuvette (taking care not to pick-up microspheres from the bottom of the tube). It may be necessary to remove some samples from the cuvette and dilute DMF in order to keep the absorbance readings in the linear region. Absorbance readings should made be below 1.5 AU in order to keep the measurements in the linear region of dye analysis.
OPTION 2: Insert-Filter Recovery of Microspheres and Dye
13.) Place a new BD Cell Culture Insert Filter (see Materials List) into the top of a 15mL 'Waste Collection' centrifuge tube labeled with Sample Identification. The insert will fit inside the centrifuge tube with the filter ring resting on top of the centrifuge tube. The ‘Waste Collection' centrifuge tube can be a 'used-tube', as the contents will be later discarded. Transfer the microsphere sample with a 200uL disposable-tip pipette onto the top of the filter cup (see Note 2. below). Rinse the originating centrifuge sample tube with approximately 100uL of DMF from a wash-bottle, vortex-mix and transfer to the filter. Repeat the rinse a second time to insure the complete transfer of microspheres. Any samples which were split into multiple digestion tubes should now be recombined into a single sample on top of filter insert. Rinse as described above.
14.) Centrifuge the 'Waste Collection' tubes with filter for 5 min. at 1,500g. The filter will retain the colored microspheres and a very small amount of tissue debris. All of the aqueous waste solution will be collected at the bottom of the centrifuge tube
15.) Carefully remove each filter-insert from the waste collection centrifuge tubes and place each into the top of a new 15mL ‘Dye Collection’ centrifuge tube and transfer the Sample Identification to the new tube. Add 200uL DMF to the filter cup. Let the DMF stand on top of each filter for 15 m in to allow the DMF to completely extract the colored dye from the microspheres.
16.) Centrifuge the new Dye Collection tubes with filter for 5 min. at 1,500g. Each centrifuge tube will collect the dye eluded by DMF. Retain all the filter-inserts (with Sample Identification) until spectrophotometric analysis has confirmed positive microsphere recovery, and then discard all the filter inserts. It is recommended to place the filter inserts on the top of the appropriate waste collection tube to maintain filter identification.
17.) Prior to the analysis of the DMF elution samples, perform a baseline spectrophotometric absorbance run with clear DMF across the entire wavelength spectrum of interest.
Analyze each DMF sample by absorbance spectrophotometry using an appropriate volume microcuvette (100 to 200ul). It may be necessary to remove some samples from the cuvette and dilute with DMF in order to keep the absorbance readings in the linear region. Absorbance readings should be made below 1.5 AU for the highest absorbance peak in order to keep the measurements in the linear region of dye analysis.
HINTS AND NOTES:
This section describes the quantification of blood flow derived from the dye absorbance measurements described in the preceding section. This section assumes that the dye samples have been diluted to achieve the proper absorbance values below 1.3 AU.
Computing regional blood flow from local injection, corrected for wet
weight is achieved by the following equation:
[AU per sample] [Total AU]
------------------------- = --------------------- {1}
[flow to the sample] [Measured inflow]
rearranging {1};
[AU per sample][Measured inflow]
[flow to the sample] = ----------------------------------- {2}
[Total AU]
Computing regional blood flow for a system injection corrected for
wet weight is achieved by the following equation:
[AU per sample] [AU per reference sample]
------------------------ = ------------------------------- {3}
[flow to the sample] [Reference withdrawal rate]
rearranging {3};
[AU per sample][Reference withdrawal rate]
[flow to the sample] = ----------------------------------------------- {4}
[AU per reference sample]
In a manner similar to the overlap correction in the counting of radioactive
microspheres, the composite spectrum of dye solutions can be resolved into
spectra of single constituents by a matrix inversion technique (7).
It is also possible to compute cardiac output from Dye-Trak®
microspheres measurements. This requires careful determination of the dose,
as well as spectral quantification of residual microspheres which remain in
the injection syringe. Cardiac output is defined as:
[Reference Flow x Total AU]
CO = ---------------------------------- {5}
[AU per reference sample]
in which the Injectate AU is equal to the Total AU minus the Residuals
trapped in the injection syringe.
I. Product Identification
PRODUCT: Colored Particle Size Standards
CONTENTS: Aqueous Suspensions of Polymer Microspheres containing colored Organic Dye. Polymers include: Polystyrene, Polystyrene Divinylbenzene, and Polymethylstyrene.
II. Hazardous Ingredients
Typical Values: Ingredient Percent Hazard Data Solid Plastic Spheres 0.05-2.0 Non-hazardous Water 89-99.95 Non-hazardous Polyoxyethylenesorbitan 0.01 Non-hazardous Thimerosal 0.01 Non-hazardous Organic Dye <0.005 Non-hazardous
III. Physical Data
Boiling Point: 100 C
Vapor Density: NA
Volatiles, percent by volume: 89-99.95% as water
Water Solubility: Material is a suspension of insoluble colored plastic spheres in water.
Specific Gravity: 1.0-1.09
Molecular Weight: Varies
Appearance: Colored liquid
IV. Health Hazard Data
EFFECTS OF EXPOSURE:
Eyes: Possible minor irritation.
Skin: Non-irritating, except for prolonged and repeated exposure.
Ingestion: Possible minor gastric irritation.
Inhalation: No known adverse effects, but respiration of fine particles should be avoided as a general principle.
FIRST AID:
Eye Contact: Wash thoroughly with water. If irritation persists, consult a physician and stain for corneal abrasion.
Skin Contact: Wash thoroughly with soap and water.
Inhalation: Remove to clean air. Consult a physician if irritation persists.
Ingestion: For large volumes, induce vomiting and consult a physician.
V. Fire and Explosion Hazard Information
The suspensions are non-flammable. For dried material, use CO2, water or dry chemical extinguisher. Combustion may produce noxious gases. Use suitable breathing equipment.
VI. Reactivity Data
Stability: The suspensions are chemically stable and no polymerizations will occur. They are incompatible with highly ionic solutions, such solutions will cause the particles to flocculate.
VII. Storage and Handling Precautions
Avoid creating, ingesting or inhaling dusts or aerosols of fine particles. Keep tightly sealed to prevent contamination. Avoid damaging or puncturing containers. Store between 4C and 25 C. DO NOT FREEZE.
VIII. Special Protection Information
Respiratory Protection: None required under normal usage. Filtered respirator is recommended for aerosol or dust production.
Ventilation: Normal ventilation is sufficient for most applications.
Gloves: Recommended.
Eye Protection: Highly recommended.
Protective clothing: Recommended.
IX. Spill, Leak and Disposal Procedures
Spills: AVOID CREATING, INGESTING OR INHALING DUSTS OR AEROSOLS. Wipe or wash up material. Caution: Surfaces covered with dried microspheres may become slippery.
Disposal: Small volumes (0.1 liter or less) may be disposed of in laboratory sinks and drains. Flush with water. Material may clog drains in large quantities. Bury large amounts of dried material in approved landfill.
Triton Technology utilizes precision polystyrene microspheres with a 15 micrometer nominal diameter and a standard deviation of 0.2 micrometers. Actual average diameter and standard deviation for each batch is recorded during our quality assurance procedure. The batch number is printed on the label of each one of our bottles.
White spheres, part #155-0370, are dyed with Blankophor MAN from Bayer. The typical absorbance for dye extracted from 1000 of these spheres is .059 AUs in 100 ul. of DMF. In addition, these microspheres fluoresce when excited with ultraviolet (300- 400 nm) energy, emitting blue (400-450 nm) energy.
Yellow spheres, part #155-0448, are dyed with Bayer Resolin Brilliant Gelb. The typical absorbance for dye extracted from 1000 of these spheres is .068 AUs in 100 ul. of DMF. In addition, these microspheres fluoresce when excited with blue (400- 480 nm) energy, emitting green (500-600 nm) energy.
Red spheres, part #155-A530, are dyed with Terasil-Rot manufactured by CIBA. The typical absorbance for dye extracted from 1000 of these spheres is .18 AUs in 100 ul. of DMF.
Violet spheres, part #155-0594, are dyed with Resolin Brilliant Rotviolett FBL from Bayer. The typical absorbance for dye extracted from 1000 of these spheres is .032 AUs in 100 ul. of DMF.
Blue spheres, part #155-0672, are dyed with Resolin Brilliant Blau BGLN from Bayer. The typical absorbance for dye extracted from 1000 of these spheres is .045 AUs in 100 ul. of DMF.
The following suppliers carry the equipment, supplies and reagents necessary for handling and processing Dye-Trak® microspheres. The list is provided as a guide, use your own judgment in dealing with these sources.
Beckman Instruments (Spectrophotometers)
Mail Station D-33-D
2500 Harbor Boulevard, Box 3100
Fullerton, CA 92834
Telephone:(714)871-4848
Fisher Scientific (Reagents, Glassware, General Supplies)
711 Forbes Avenue
Pittsburgh, PA 15219
Telephone:(412)490-8300
Harvard Apparatus (Syringes, Peristaltic Pumps)
22 Pleasant Street
South Natick, MA 01760
Telephone: (508)655-7000
Toll Free in US and Canada:(800)272-2775
Hellma Scientific Cells (Spectrophotometer Cuvettes and Related Equipment)
Box 544 Borough Hall Station
Jamaica, NY 11424
Telephone:(718)544-9534
FAX:(718)263-6910
Hewlett-Packard (Spectrophotometers)
2 Choke Cherry Road
Rockville, MD
Telephone:(301)670-4300
Millipore (Filtration Equipment)
80 Ashby Road
Bedford, MA 01730
Telephone: (800)645-5476 or (617)533-8125
Fax: (800)645-5439
Tomy Tech (Centrifuges)
2452 Embarcadero Way
Palo Alto, CA 94303
Telephone: (415)424-0898
Fax: (415)424-0897
Toll Free in US and Canada:(800)545-TOMY
Triton Technology (Dye-Trak® microspheres, Filters, MISS
Software)
4616 Santa Fe Street
San Diego, CA 92109
Telephone: (619)272-1251
Fax: (619)272-1451
Toll Free in US and Canada: (800)872-1251
Sigma Chemical Company (Reagents, Glassware)
POB 14508
St. Louis, MO 63178
Telephone:(314)771-5765
FAX:(314)771-5757
Toll Free in US and Canada:(800)325-3010
Up to date references can be found in our Dye-Trak® Bibliography
1. Kowallik, P., Schulz, R., Guth, B., Schade, A., Paffhausen, W., Gross, R., Heusch, G.: Measurement of regional myocardial blood flow with multiple colored microspheres. Circulation Research 83(3):974-982,1991.
2. Rudolph, A.M., Heymann, M.A.: The circulation of the fetus in utero: Methods for studying distribution of blood flow, cardiac output and organ blood flow. Circulation Research 21:163-184,1967.
3. Makowski, E.L., Meschia, G., Droegemueller, W., Battaglia, F.C.: Measurement of umbilical arterial blood flow to the sheep placenta and fetus in utero: Distribution to cotyledons and the intercotyledonary chorion. Circulation Research 23:623-631,1968.
4. Domenech, R.F., Hoffman, J., Noble, M., Saunders, K.B., Henson, J.R., Subijanto, S.: Total and regional coronary blood flow measured by radioactive microspheres in conscious and anesthetized dogs. Circulation Research 25:581-596,1969.
5. Hale, S.L., Alker, K.J., Kloner, R.A.: Evaluation of nonradioactive, colored microspheres for measurement of regional myocardial blood flow in dogs. Circulation 78:428-434,1988.
6. Morita, Y., Payne, B.D., Aldea, G.S., McWattes, C., Huseini, W., Mori, H., Hoffman J., Kaufmann, L.: Local blood flow measured by fluorescence excitation of nonradioactive microspheres. Am J Physiol 258:H1573-H1584,1990.
7. Schosser, R., Arfors, K-E., Messmer, K.: MIC-II - A program for the determination of cardiac output, arterio-venous shunt and regional blood flow using the radioactive microsphere method. Comput Programs Biomed 9:19-38,1979.
8. Baer, R.W., Payne, B.D., Verrier, E.D., Vlahakes, G.J., Molodowitch, D., Uhlig, P.N., Hoffman, J.: Increased number of myocardial blood flow measurements with radionuclide-labeled microspheres. Am J Physiol 246:H418-H434,1984.
9. Sasayama, S., Franklin, D., Ross, J. Jr., Kemper, W.S., McKown, D.: Dynamic changes in left ventricular wall thickness and their use in analyzing cardiac function in the conscious dog. Am J Cardiol 38:870-879,1976.
10. Schulz, R., Hucking, G., Heusch, G.: "CORDAT"-a new data acquisition and reduction program. Eur Heart J 10:309 (abstr.),1989.
11. Schulz, R., Miyazaki, S., Miller, M., Thaulow, E., Heusch, G., Ross, J., Jr., Guth, B.D.: Consequences of regional inotropic stimulation of ischemic myocardium on regional myocardial blood flow and function in anesthetized swine. Circ Res 1989;64:1116-1126
13. Utley, J., Carlson, E.L., Hoffman, J., Martinez, H.M., Buckberg, G.D.: Total and regional myocardial blood flow measurements with 25u, 15u, 9u, and filtered 1-10u diameter microspheres and antipyrine in dogs and sheep. Circulation Research 34:391-404,1974.
14. Fan, F.C., Schuessler, G.B., Chen, R.Y.Z., Chien, S.: Determinations of blood flow and shunting of 9- and 15-um spheres in regional beds. Am J Physiol 237:H25-H33,1979.
15. von Ritter, C., Hinder, R.A., Womack, W., Bauerfeind, P., Fimmel, C.J., Kvietys, P.R., Granger, D.N., Blum, A.L.: Microsphere estimates of blood flow: methodological considerations. Am J Physiol 254:G275-G279,1988.
16. Gallagher, K.P., Kumada, T., Koziol, J.A., McKown, M.D., Kemper, W.S., Ross, J., Jr.: Significance of regional wall thickening abnormalities relative to transmural myocardial perfusion in anesthetized dogs. Circulation 62:1266-1274,1980.
17. Phibbs, R.H., Wyler, F., Neutze, J.: Rheology of microspheres injected into circulation of rabbits. Nature 216:1339-1340,1967.
18. Phibbs, R.H., Dong, L.: Nonuniform distribution of microspheres in blood flowing through a medium-size artery. Can J Physiol Pharmacol 48:415-421,1970.
19. Reed, J.H., Jr., Wood, E.A.: Effect of body position on vertical distribution of pulmonary blood flow. J Appl Physiol 28:1970
20. Millard, R.W., Baig, H., Vatner, S.F.:Cardiovascular effects of radioactive microsphere suspensions and tween 80 solutions. American Journal of Physiology V 232:H331-H334,1977.
21. Hendrik, E.E., van 't Veen, A., Gommers, D., Segerer, H., Lachmann, B.: Colored microspheres versus radioactive labeled DPPC in a pulmonary distribution study of surfactant in rabbits. Euro Wkshop on Flourescent Microsphere Method Naastricht,1993.
22. Baumgart, D., Ehring, T., Heusch, G.: Proischaemic action of nisoldipine: Relationship to a decrease in perfusion pressure and comparison to dipyridamole. Cardiovascular Research 27:000-000,1993.
23. Baumgart, D., Ehring, T., Kowallik, P., Guth, B.D., Krajar, M., Heusch, G.: Impact of -adrenergic coronary vascoconstriction on the transmural myocardial blood flow distribution during humoral and neural adrenergic activation. Circulation Research 73:869-886,1993.
24. Kolok, A.S., Spooner, R.M., Farrell, A.P.: The effect of exercise on the cardiac output and blood flow distribution of the large scale sucker catostomus macrocheilus. J Exp Biol 183:301-321,1993.
25. Sidi, A., Rush, W.: Low correlation between radioactive and multiple colored microspheres used to measure regional myocardial blood flow when flow is low or high. SCA 15th Annual Meeting 338,1993.
NUMBER OF MICROSPHERES PER ml
Number of Particles/ml = (Solid Content %) 1.910 x 10tenth power
(Dcubed) (density)
where D = Diameter in m
where density = density of polymer in grams/ml
For Polystyrene, Polystyrene/DVB, density = 1.050 gram/ml
= (Solids Content %) 1.819 x 10tenth power
Dcubed
Example: Solids Content = 10% for 15 m : 53.90 x 10sixth power/ml
NUMBER OF MICROSPHERES PER GRAM
Number of Particles/Gram = 1.910 x 10twelfth power
(Dcubed) (density)
where D = Diameter in m
where density = density of polymer in grams/ml
For Polystyrene, Polystyrene/DVB, = 1.050 gram/ml
= 1.819 x 10twelfth power
Dcubed
Example: 15 m : 539.0 x 10sixth power/gram
Copyright 1995 Triton Technology, Inc.
Updated May 29, 1998