The following protocol is used in the laboratory of Professor John Wingfield at the University of Washington, Seattle. It is meant to be used under supervision in this lab. Normally it may take several weeks or even months to become proficient with the assay techniques. The Wingfield Lab is not responsible for consequences occurring when persons attempt to use this protocol on their own.
Plasma concentrations of several steroids may be determined from a single, relatively small (50-200µl) plasma sample by performing a radioimmunoassay. Steroids are first extracted from the plasma using dichloromethane. This extract is then evaporated and dissolved in ethyl acetate/iso-octane and placed on celite columns. Steroids are separated from one another by partition chromatography. This is done by passing increasingly polar mixtures through the columns, with one steroid coming off with each fraction. The separated steroids are then assayed with a labelled steroid and an antiserum. By generating a standard curve with known amounts of steroid it is possible to calculate the percentage of labelled steroid that binds to the antiserum, and thus inversely calculate the unknown amount of steroid in the plasma.
The general procedure for accomplishing the radioimmunoassay is described below. Variations are described in the appendices. Abbreviations for steroids are P (progesterone), DHT (dihydrotestosterone), T (testosterone), E2 (estradiol), and B (corticosterone). Other abbreviations are HS (Hamilton syringe), EP (Eppendorf pipette), and ER (Eppendorf repeating pipette).
SOLVENT DISTILLATION ^ Back to top
We purchase ACS reagent quality dichloromethane and ethyl acetate. These are distilled within 24 hours of use, using a standard distillation apparatus (distillation flask, condenser, heater, collection vessel). The first and last 50 ml are discarded. For dichloromethane, a Variac controlling the heater is set at 45. For ethyl acetate it is set at 70-75. Should you accidentally distill the entire amount (fail to leave the last 50 ml), you should start the process over. Iso-octane (2,2,4 trimethylpentane) cannot be distilled due to extreme flammability, so we use nanograde quality only (Mallinckrodt).
PREPARATION OF PLASMA SAMPLES Day 1 ^ Back to top
Set up plasma samples in glass centrifuge tubes (pointed bottoms, 12 ml). The first and last tubes are always blanks and contain only distilled water (dH2O). The second tube is always a standard into which measured amounts of the steroids being assayed are placed. This measures the accuracy of the current assay as well as interassay variation, when samples may be spread over more than one assay. When all plasma samples have been measured, a small amount of labelled steroid (20 µl of each steroid being tested=2,000cpm) is added to all tubes except the blanks. Total cpm is measured at the end to determine what percentage of the labelled steroid is recovered, and, assuming that the unknown steroid behaves the same, the final dose of the unknown steroid is adjusted according to this recovery value.
Setting up plasma samples. Set up a data sheet for plasma volumes, numbering samples as follows: 1. blank; 2. standard; 3....samples; last = blank. Mix plasma on a vortex mixer if you have more than needed. Measure 100-400 µl plasma, using HS, for each sample and place in centrifuge tube. 100-200 µl is ideal. Rinse syringe with dH2O and add to plasma. Bring all tubes to the same volume by rinsing the syringe (usually 100 or 200 µl). When all tubes are completed, go back and bring all volumes to 400 µl using the ER. Note: If LH will be performed on the same samples, save 25 µl plasma and refreeze).
Setting up standard tube. The second assay tube is the standard. Place 50 µl of cold (non-radioactive) steroid of each steroid being assayed into this tube. This will be 500 pg P, 250 pg T, E2, and DHT, and/or 1000 pg B. Bring the volume to 400 µl with dH2O.
Setting up the blanks. Add 400 µl dH2O to first and last tubes. Do not add hot steroids to these blanks.
Setting up the recoveries. For each steroid being assayed add 20 µl hot steroid to all tubes except the blanks, using either the HS or ER. Also, for each steroid add 20 µl hot steroid to a scintillation vial (one vial for each steroid). Cap, label cap, add scintillant, and store in a dark place. These vials will measure the total cpm to determine the percentage recovered.
Vortex all tubes and refrigerate overnight at 4oC to allow hot steroids to equilibrate with plasma lipids and binding proteins.
EXTRACTION OF STEROIDS AND COLUMN PACKING Day 2 ^ Back to top
Add 5 mls distilled dichloromethane to each sample. Vortex each sample at a low speed, wearing protective gloves and clothing. Let tubes stand for at least 2 hours. Progesterone must be extracted with ethyl ether - see Appendix V.
Packing Celite Columns. Celite (diatomaceous earth) should be heated at 1000oF for 24 hours prior to use. Range must be 950-1100oF, so oven gauge must be accurate. Remove from oven (with great care!) and allow to cool for about 45 minutes prior to weighing. Cooling can occur while dichloromethane is being added to samples.
Water trap. Mix 6 gms celite with 2 mls dH2O using mortar and pestle. Mix well and then mix some more. Place a #2 glass bead in the bottom of a 5 ml disposable pipet. Put a rubber band around the pipet to hold it in the rack. Position band around the 1.5 mark. Shake celite mix into the column, using a funnel, to about the 3.5 mark. Using a glass rod, pack firmly to the 4.5 mark. Generally, the celite mix packs to about 1/3 of the loose volume. Pack all water traps. Cover and save any leftover celite mix in case columns get broken during next phase and have to be redone.
Glycol phase. Mix propylene glycol (1,2 propandediol) and ethylene glycol in equal amounts. Add 3 mls of glycol mix to 6 gms celite and mix with a mortar and pestle until homogeneous. Add mix to the 3 mark on the column and pack down to 4. Add again to the 2.5 mark and pack to 3.5. Add once more to the 2 ml mark and pack down to 3. The columns are now packed with 1.5 ml celite/glycol mixture on top of 0.5 ml celite/water mixture. (See Appendix II for short column procedures).
Wetting the columns. Add 4 mls pure (nanograde) iso-octane to each column using a glass syringe. Attach hoses and allow nitrogen to flow through the columns. Stop when solvent level drops to within 1 mark of the celite. Add another 4 mls iso-octane to each column, but this time bring solvent just into celite. Nitrogen pressure may be as great as desired during this phase, but be careful not to dry out the celite. Should this occur, it must be rewet with enough iso-octane to wet it thoroughly. Columns that dry out badly will never run as rapidly as those not dried out.
Extraction Of Samples. While the columns are getting wet, aspirate the dichloromethane and extracted steroids (lower phase) with a disposable Pasteur pipet. Use a new pipet for each sample, and wear gloves. Gently blow bubbles through the plasma layer to avoid getting plasma into the pipet. Remove as much dichloromethane as possible without getting any plasma. Place dichloromethane into a 13x100 mm test tube, one tube for each sample. Place these tubes into a rack which fits the nitrogen evaporator. Evaporate the dichloromethane using the evaporator with the tubes in a water bath at 40oC.
COLUMN CHROMATOGRAPHY Day 2-3 ^ Back to top
Adding samples to columns. Using EP, add 0.5 ml of 10% ethyl acetate in iso-octane to each sample. Vortex first tube, and add contents to first column with a disposable pipet. Add another 0.5 ml of 10% mix to test tube, voretx and add to column (2nd time is to rinse). Leave pipet stuck in column to mark your place and to avoid adding two samples to the same column. Proceed to the next sample. When all have been placed on columns remove and discard the pipets.
Attach hoses to columns and turn on nitrogen. Regulate the pressure so that none drips faster than 1 drip every 6 seconds. When the solvent reaches the top of the celite turn off that column and remove the hose. Do not dry out. Discard this eluate using methods approved by your institution. Columns may now be left overnight at any point after steroids have been added. To do this add the appropriate ethyl acetate/iso-octane mix, attach hoses, turn on nitrogen to get things started, then turn off nitrogen at tank and allow a slow drip overnight. They will not run dry provided nitrogen was turned off at tank. When running the following fractions, collect only those wanted, but all fractions must be run unless noted below. Collect into 13x100 mm test tubes and place in a rack for evaporation.
Add 4 mls pure iso-octane and blow down under regulated pressure as above. The eluate is P. However, if P will be collected, use 4 mls of 2% ethyl acetate in iso-octane instead of pure iso-octane.
Add 4.5 mls 10% ethyl acetate in iso-octane as above. The eluate is DHT.
Add 4.5 mls 20% ethyl acetate in iso-octane. The eluate is T.
Add 4.5 mls 40% ethyl acetate in iso-octane. The eluate is E2. This step is optional and done only when assaying for E2.
Add 4.0 mls 50% ethyl acetate in iso-octane (4.5 mls if you skipped the 40% fraction). The eluate is B.
Dry purified extracts under nitrogen evaporator in a 40oC water bath until totally evaporated. Add 550 µl buffer (PBSG) to each tube for all except B. Add 1 ml buffer to B tubes. Buffer can be added using a combination of tips on the ER or with a variable pipet. Vortex, cover (with foil or parafilm), and refrigerate overnight. Note: You may place rack on shaker and shake for 45 minutes and proceed to set up curves.
RADIOIMMUNOASSAY Day 4 ^ Back to top
Set up the appropriate number of small (10x75 mm), disposable test tubes in a rack. First arrange 12 pairs for the standard curve, with a space between the 3rd and 4th pair. Follow with a pair of tubes for each sample. If you have more than one spin of the centrifuge it is best to have each spin set up individually in racks.
For all steroids except corticosterone:
Setting up the samples. Vortex each tube before partitioning. Using an EP, pipet 200 µl of each sample into duplicate assay tubes. Either simultaneously or following, pipet 100 µl into a scintillation vial for the recovery. Scintillant will then be added to these vials. Cap and vortex.
Setting up the standard curve: The standard curve will be used to determine the dose of steroid in the unknown samples. The first three tubes are B1-B3. B1 measures total cpm, B2 non-specific binding (or background), and B3 maximum binding with the antiserum. The remaining 9 pairs of tubes (S1-S9) generate the curve.
Add 200 µl buffer to the B1 and B2 tubes, 100 µl buffer to remaining tubes except for S1, using the ER (5 ml tip set on 1). Add 100 µl cold steroid to each of the S1 and S2 pairs, using the HS. This will be 1000 pg for P, 500 pg for DHT, T, and E2, and 2000 pg for B. Vortex the S2 tubes, remove 100 µl and add to the S3 tubes. By continuing this serial dilution the concentration will be halved each time. Discard the last 100µl removed from the S9's. Go back and add 100µl buffer to B1-B3 and S1-S9 tubes (to bring up to same volume as samples). Gently mix (do not shake) hot steroid and antiserum prior to use. Using the ER, add 100 µl hot steroid to all tubes (standard curve plus unknown samples). Add 100 µl antiserum starting with the B3 tubes to all tubes (no antiserum in B1 and B2). Mix on the rack shaker and refrigerate overnight. Curves may be left in the refrigerator longer before adding charcoal, but overnight is the minimum time.
The standard curve contains:
Note: When more than 48 samples occur, extra spins of the centrifuge are required. To correct for variations between spins, a new B2 and B3 are set up for each spin of the centrifuge.
Setting up corticosterone samples and curve:
The procedure for B is the same except different amounts are used.
Setting up the samples: Pipet 100 µl unknown sample into pairs of test tubes for radioimmunoassay. Pipet 200 µl unknown sample into a 7 ml scintillation vial to measure the recovery. This is the reverse of the procedure for all other steroids.
Setting up the standard curve: The procedure is identical except do not add the last 100 µl buffer to the B and S tubes following the serial dilution. This is because the unknowns only have 100 µl, so the volumes in the curve and the samples are identical. Otherwise, all instructions above apply
SEPARATION OF BOUND AND FREE COUNTS Day 5 ^ Back to top
Dextran-coated charcoal will be added to all tubes (except B1) and allowed to adsorb all unbound steroid. Tubes will be centrifuged, and the supernatant will be decanted into 7 ml scintillation vials for counting cpm.
CLEAN-UP ^ Back to top
Glassware should be cleaned daily throughout the assay. Items are rinsed with tap H2O, soaked in a solution of Count-Off (from NEN) overnight, rinsed 3 times in tap H2O, then either submersed or rinsed 2 times in dH2O. Columns must be soaked before the celite can be removed (back pressure from a faucet attachment or a pipet washer) and then soaked in Count-Off. After final rinse, glassware is placed in a drying oven set below 40oC. If the temperature goes higher, absorption sites are set up on the glassware, causing assay problems. Should this occur, re-wash and dry.
Scintillation vials containing radioactive
waste must be properly discarded. Follow guidelines established
by your institution. For tritium waste, we empty the vials into
gallon jugs, discard the caps after rinsing, wash the vials in
Count-Off, and then dry and box for reuse or discard with regular
glass garbage. The gallon jugs are cheaper to dispose of than
the full vials. We do this even though we use a biodegradable
scintillant (Ultima Gold - Packard Instrument Co.). Regulations
regarding putting biodegradable scintillants down the sink vary
from one institution to another. You must check with your own
institution about proper disposal.
Each person is responsible for disposing of his/her vials in a timely manner. This should be immediately following a successful run on the scintillation counter and reading results. If all vials have counted then they can be discarded.
APPENDIX I - DIRECT ASSAYS ^ Back to top
When separation of steroids in a plasma sample is unnecessary, a direct assay may be performed without columns. Generally this is only done with corticosterone (B), estradiol (E2), and progesterone (P). Other steroids have cross-reactions with the antisera and must first be separated from each other.
To perform a direct assay for corticosterone, E2 or T/DHT:
To perform a direct assay for E2 or P:M
"Little B Assays" ^ Back to top
When assaying for B and you have very small plasma volumes, you can do a "little B assay." The procedure is the same as for corticosterone above except:
APPENDIX II - SHORT COLUMN CHROMATOGRAPHY ^ Back to top
Short columns may be used if not assaying for both B and E2. The differences from a regular assay are:
Fractions: For P, DHT, T, B.
When samples have been added to columns and blown down into celite with regulated nitrogen pressure:
For P, DHT, T, and E2:
APPENDIX III - CALCULATIONS
Unless you have access to a scintillation counter that calculates and draws the standard curve and then calculates doses, you must generate the curve and then estimate doses from the curve for your unknown samples.
To generate the curve:
Average the values from the pairs of B2's-S9's. Subtract the B2 value from the B3-S9. Divide each new S value by the new B3 value. This is the % bound.
% bound = Sx-B2
For each S value, draw the % bound on semi-log
paper and draw the curve.
Average the values from the pairs of your unknown samples and calculate the % bound exactly as you did for the S1-S9. Plug this value into the curve and estimate pg of steroid in your sample.
To calculate final plasma concentration of steroid
For P, DHT, T, E2, and "little B":
value from curve (pg) x 2.75 1000
Plasma concentration = ______________________ X ________
(pg/ml) % recovery (decimal) * µl plasma
Estimated dose from curve = 30 pg
Recovery = 0.764
Plasma volume = 150µl
30 x 2.75 1000
Plasma concentration = _______ X ____ = 719.9 pg/ml
For B (done in the usual manner, sometimes called "big B')
value from curve (pg) x 10 1000
plasma concentration = _____________________ X ________
(pg/ml) % recovery (decimal) µl plasma
Calculation of recoveries:
For P, DHT, T, E2, little B (when recovery
Divide total cpm (taken from vial with 20µl of hot steroid) by 5.5. Divide all cpm from recoveries by this value.
For B (big B):
Divide total cpm by 5. Divide all cpm from recoveries by this value.
APPENDIX IV - REAGENTS ^ Back to top
Steroid Assay Buffer - PBSG
(0.1M, pH 7.0)
NaH2PO4 : H2O 16.14 gm
Na2HPO4 : 7H2O 49.05 gm
NaCl 27.00 gm
gelatin 3.00 gm
NaN3 3.00 gm
dH2O 3 L
Mix in 4 L jug, and place on heated, magnetic stirrer. Heat over low heat while stirring until gelatin is dissolved. Refrigerate.
Charcoal 2.5 gm
Dextran T-70 0.25 gm
PBSG 1 L
Place charcoal and dextran in bottle. Add PBSG and shake well. Refrigerate.
We use Ultima Gold, a biodegradable, single
phase scintillant from Packard Instrument Co. (800-323-1891).
It can handle a 20% aqueous load without forming precipitates.
APPENDIX V - PROGESTERONE EXTRACTION ^ Back to top
To extract progesterone, you must use ethyl ether (we use Mallinckrodt brand). Add 4 mls ethyl ether to each centrifuge tube in the hood. Vortex at a low speed. Let sit for 10 minutes. Have a Dewar's thermos with dry ice and methanol or acetone ready. Dip the tip of each centrifuge tube into this cold liquid for about 15 seconds, freezing the plasma. Decant the ether extract into 13x100 mm test tubes and place in a rack for the evaporator. When all tubes have been done, place the rack in the H2O bath at room temperature and evaporate under nitrogen.
If cortisol is not being determined with progesterone, the ether extraction will be sufficient. Ethyl ether extracts all steroids except cortisol.
When running fractions of ethyl acetate in
iso-octane on the columns, after the samples have been added to
the columns and blown down, add 4 mls of 2% ethyl acetate in iso-octane
instead of 4 mls of pure iso-octane. This brings most of the progesterone
off in this fraction. Otherwise it tends to come off in both the
0% and the 10% fractions.
RIA SHORT SHEET ^ Back to top
Measure plasma samples into centrifuge tubes. Set up blanks and
standard tube (50 µl cold steroid for each steroid being
Add 20 µl hot steroid to all tubes except blanks and a scintillation vial (for each steroid being assayed).
Bring all volumes to 400 µl with dH2O.
Let equilibrate overnight in refrigerator.
Add 5 ml dichloromethane and vortex.
Let sit > 2 hours.
Columns: Heat celite > 24 hours at 1000°F.
Cool and weigh.
Water trap (6 gm:2 ml). Pack to 4.5 mark.
Glycol phase (6 gm:3 ml of 1:1 glycol mix). Pack to 3 mark in 0.5 ml increments.
Wet columns with iso-octane.
Extraction: Aspirate dichloromethane (lower phase) with disposable pipet into 13x100 mm test tubes in rack for evaporator. Evaporate under nitrogen in 40°C water bath.
Add samples to columns. Add 0.5 mls 10% ethyl acetate in iso-octane to tubes, whirl mix, pipet onto columns with disposable pipet. Rinse with another 0.5 mls and add to column. Run liquid into celite with regulated nitrogen pressure no faster than 1 drip/6 seconds.
Partition chromatography: Add appropriate fractions of ethyl acetate in iso-octane under regulated nitrogen pressure and collect or discard eluate:
0% 4.0 mls P (2% if collecting P)
10% 4.5 mls DHT
20% 4.5 mls T
40% 4.5 mls E2
50% 4.0 mls B
Evaporate liquid and add buffer: Evaporate
under nitrogen in 40°C H2O bath.
Add 550µl buffer to all but B, 1000 µl to B. Let sit overnight at 4°C.
Partitioning samples: For all except B: place 200µl in paired sample tubes, 100µl in scintillation vials for recoveries. For B place 100µl in paired sample tubes, 200µl in recoveries.
Standard curve: Refer to procedure. Refrigerate overnight.
Separation and counting: Add 0.5 mls charcoal
(H2O to B1). Let sit 12 minutes. Load centrifuge while waiting.
Centrifuge for 10 minutes at 2000 rpm.
Decant into scintillation vials.
Add scintillant, cap, mix.
Count on scintillation counter.
Clean glassware, empty vials and dispose of
radioactive waste. Be a good citizen or at least not a turkey!
PREPARATION OF ANTIBODY ^ Back to top
is purchased from Wien Laboratories, P. O. Box 227, Succasunna,
NJ 07876, Phone 800-631-9384. Catalog number is T-3003 and costs
about $20/vial. Each vial makes 100 ml. Store vials frozen (-20oC).
Thaw vial. Measure 5 ml PBSG. Pipet into the vial of T antiserum and swirl gently. Let this set in the refrigerator overnight. The following day pipet this into a bottle. Continue to add 95 mls PBSG (rinse vial and pipet it out into the bottle several more times). Add the remainder of the PBSG to the bottle and swirl to mix. Never shake antisera vigorously. Maximum binding is usually around 20-25%.
Progesterone: Also purchased
from Wien Laboratories (as for T/DHT). Catalog number is P-1604
and also costs about $20/vial. Each vial makes 50 ml. Store vials
Measure 5 ml PBSG and follow procedure for T antibody, using 50 mls total PBSG instead of 100 mls. Maximum binding is usually 40-45%
Estradiol: Antibody is purchased
from Arnel, 119 Washington Place, Suite B, New York, NY 10014,
Phone 212-620-4622. Catalog number is 1702 and costs about $200/vial.
Each vial makes 1000 ml. Store vials frozen.
Thaw vial. Using a disposable pipet tip, pipet 20 µl of this solution into a volumetric flask. Bring up to 100 ml in PBSG. Swirl gently to mix. This needs to be tested with a trial standard curve for proper dilution. It is never the same twice. Maximum binding is usually 40-50%. If higher dilute accordingly.
is purchased from Endocrine Sciences, 4301 Lost Hills Road, Calabasas
Hills, CA 91301, Phone 818-880-8040. Catalog number is B3-163
and costs $100 for 1 vial and less for more vials. Each vial makes
170 ml. Store vials frozen.
Thaw vial and add 1.7 ml dH2O to the vial. Shake gently by hand to mix. You may remove 1 ml of this solution and add to 100 ml PBSG and freeze the remaining vial contents to be used later. When using the remaining 700 µl, thaw it and mix with 70 ml PBSG, rinsing vial with PBSG as for T antibody. You may mix the entire 1.7 ml into 170 mls PBSG if you are doing a lot of corticosterone assays and will use it within a few months. Maximum binding is 20-25%.
"COLD" STEROIDS - STANDARDS ^ Back to top
Stock solutions of steroids are made and kept for further dilution in the assay. These steroids are purchased from Sigma (P=P0130, DHT=A8380, T=T1500, E2=E8875, B=C2505). They are weighed out in a balance away from the assay area and which will not be used to weigh anything that comes in contact with the assay. Weigh 100 mg of steroid onto a small piece of aluminum foil. Carefully fold this up to contain the steroid and drop into a 100 ml volumetric flask. Add 100 ml pure EtOH, seal, and swirl to mix. You now have 1 mg/ml of steroid in stock solution. This is very concentrated in terms of the assay and must be handled carefully or you can contaminate the assay.
For E2 and androgens: We want a dilution to be 500 pg/100 µl, which is the same as 125 ng/25 ml. In an area away from the assay bench, using a disposable pipet tip, take 100 µl of the stock solution (= 10 µg) and dilute it into 100 mls dH20. Discard the pipet tip and all other disposables into a plastic bag for disposal. Mix this solution well. It is now 1 µg/ml = 1000 ng/ml. Using a disposable pipet tip, put 125 µl of this solution into a 25 ml volumetric flask and fill to 25 ml with PBSG. You have now achieved 500 pg/100 µl. Discard the intermediate dilution into a sink outside of the lab where you do assays, rinse the container several times, and flush the drain with water. The container can then be soaked in Count-Off.
For corticosterone: We want a dilution of 2000 pg/100 µl, which is 500 ng/25 ml. Using the same methods as above, put 100 µl of the stock solution into 100 ml dH2O and mix thoroughly. Again you have 1 µg/ml. Take 500 µl of this solution and place into a 25 ml volumetric flask. Bring up to 25 ml with PBSG. You now have 500 ng in 25 mls = 2000 pg/100 µl. Remember to use disposable tips, discard all in a plastic bag, pour the intermediate solution in a drain in another room, and rinse all glassware before bringing it back to the lab to soak.
For progesterone: By now you can extrapolate and see that you need
250 µl of the intermediate solution in 25 mls PBSG to achieve
1000 pg/100 µl.
LABEL OR TRITIATED STEROIDS
All label is ordered in 250 µCi vials from New England Nuclear Research Products, 549 Albany Street, Boston, MA 02118. They range in price from $300-$800/vial. Catalog numbers are as follows:
Testosterone [1,2,6,7,16,17-3H(N)] NET-553
Estradiol [2,4,6,7,16,17-3H(N)] NET-517
Corticosterone [1,2,6,7-3H(N)] NET-399
Progesterone [1,2,6,7,16,17-3H(N)] NET-1112
DHT [1,2,4,5,6,7-3H(N)] NET-453
Using approved safety measures for handling radioactive substances, pipet some pure EtOH into the vial, withdraw contents and place into a 10 ml volumetric flask. Continue rinsing and pipeting the solution from the vial to the flask until the vial has been rinsed several times. Bring the solution up to 10 mls with EtOH. You now have 250 µCi in 10 mls. To make solutions for the assay you want 10,000 cpm/100 µl. This works out to be about 400 µl of this stock solution (measured using a disposable pipet tip) in 100 mls PBSG, provided you rinse the small beaker you poured the stock solution into for measuring as well as the pipet tip itself. It is best to get all these extra cpm into the bottle instead of the trash or the wash, since you are paying > $1/µCi. Measure the results on a scintillation counter and adjust accordingly. It is best to add less buffer in the beginning and further dilute than to get it too weak and have to add label.
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