Multi-Species TGF-b1 LINCOplex
By purchasing this product, which contains fluorescent-labeled microsphere beads authorized by Luminex Corporation ("Luminex"), you, the customer, acquire the right under Luminex’s patent rights, if any, to use this product or any portion of this product, including without limitation the microsphere beads contained herein, only with Luminex’s laser based fluorescent analytical test instrumentation marketed under the name of Luminex*.
*Luminex instrumentation refers to Luminex®100, Luminex 200 and other Luminex instruments from MiraiBio® (MasterPlex CT™), ACS® (STarStation™), Bio-Rad Laboratories, Inc.® (Bio-Plex™) and Qiagen® (LiquiChip™). For technical assistance on running LINCOplex Kits, contact our technical service department Toll Free U.S. at 866-441-8400 or 636-441-8400 or email us at info@lincoresearch.com.
This single-plex assay kit manufactured by LINCO Research, Inc. is to be used for the quantification of human/mouse/rat/porcine TGF-ß1 in serum or tissue/cell culture medium samples. Since this kit requires sample pretreatment, TGF-ß1 cannot be multiplexed with other analytes.
This kit is for research purposes only.
A. Capture Antibody-Immobilized Bead (1X concentration): No dilution is required before use.
#73-TGF-ß1 Beads
Quantity: 1 vial containing 3.5 ml antibody-immobilized beads
B. TGF-ß1 Standard
1 vial containing TGF-ß1standard, lyophilized
200 ng/mL when reconsitituted with 250 µl water
Quantity: 1 vial
C. TGF-ß1 Quality Controls
Control I – 1 vial containing TGF-ß1, lyophilized
Control II – 1 vial containing TGF-ß1, lyophilized
Quantity: 1 vial/each Control
D. TGF-ß1 Detection Antibody
1 bottle containing biotinylated detection antibody prepared in Assay Buffer
Quantity: 3.2 ml/bottle
E. Streptavidin-Phycoerythrin
1 bottle containing Streptavidin-Phycoerythrin prepared in Assay Buffer
Quantity: 5.5 ml/bottle
F. Assay Buffer
PBS with 0.08% Sodium Azide and BSA, pH 7.6
Quantity: 2 bottles, 30 ml/bottle
G. 10X Wash Buffer
1 bottle containing 1:10 dilution required with deionized water to give 10 mM PBS with 0.05% Proclin, and 0.05% Tween 20, pH 7.4.
Quantity: 30 ml/bottle
H. Serum Matrix, lyophilized (optional - for serum/plasma samples)
Serum containing 0.08% Sodium Azide
Quantity: 1 vial, See Section VIII.E. for detailed information for reconstitution of the Serum Matrix.
I. Sample Diluent
PBS based buffer
Quantity: 5.0 ml/bottle
J. 1.0 N HCl solution
Quantity: 1.0 ml/bottle
K. 1.0 N NaOH
Quantity: 1.0 ml/bottle
L. Microtiter Filter Plate
Quantity: 1 96-Well Filtration Plate
M. Plate Sealers
Quantity: 2 Plate Sealers
III. STORAGE CONDITIONS UPON RECEIPT
Recommended storage for kit components is 2 - 8°C. See individual vials for long-term storage recommendations.
The serum matrix, once reconstituted, should be stored at –20°C.
Once the standard and controls have been reconstituted, transfer contents into polypropylene vials. DO NOT STORE RECONSTITUTED STANDARDS OR CONTROLS IN GLASS VIALS. For long-term storage, freeze reconstituted standards and controls at £ -20°C.
DO NOT FREEZE Antibody-Immobilized Beads, Detection Antibody, and Streptavidin-Phycoerythrin.
Sodium azide has been added to some reagents as a preservative. Although the concentrations are low, sodium azide may react with lead and copper plumbing to form highly explosive metal azides. Flush with a large volume of water to prevent azide build-up.
V. MATERIALS REQUIRED BUT NOT PROVIDED
A. Reagents
Luminex Sheath Fluid (Luminex Catalogue #40-50000)
B. Instrumentation/Materials
1. Adjustable Pipettors with Tips capable of delivering 25 m l to 1000 m l
2. Multichannel Pipettor capable of delivering 25 m l to 200 m l
3. Reagent Reservoirs
4. Polypropylene Microfuge Tubes
5. Aluminum Foil
6. Absorbent Pads
7. Laboratory Vortex
8. Sonicator
9. Titer Plate Shaker
10. Vacuum Filtration Unit (Millipore Vacuum Manifold Catalogue #MAVM0960R, or equivalent)
11. Luminex Instrument
VI. SPECIMEN COLLECTION AND STORAGE
A. A maximum of 25 µl of tissue culture medium or diluted serum samples can be used per well.
B. Preparation of Tissue Culture Supernatant:
Centrifuge the sample to remove cell debris and run the assays immediately or aliquot and store samples at £ -20ºC. Avoid multiple (>2) freeze/thaw cycles. Tissue culture supernatant may require a dilution with the appropriate control medium prior to assay.
See Section VIII-D for detailed sample treatment information before setting up the assay.
C. Preparation of Serum Samples (see Section VIII for sample treatment information):
Allow the blood to clot for at least 30 minutes before centrifugation for 10 minutes at 1000 x g. Remove serum and analyze immediately or aliquot and store samples at £ -20ºC. Avoid multiple (>2) freeze/thaw cycles.
See Section VIII-D for detailed sample treatment information before setting up the assay.
To obtain reliable and reproducible results, the operator should carefully read this entire manual and fully understand all aspects of each assay step before attempting to run the assay. The following notes should be reviewed and understood before the assay is set-up.
A. The Antibody-Immobilized Beads are light sensitive and must be covered with aluminum foil during all incubation times.
B. It is recommended to allow all reagents to warm to room temperature before use in the assay.
C. The bottom of the Microtiter Filter Plate should not be in contact with any absorbent material during assay set-up or incubation times. If necessary, set the plate on a plate cover or any other plate holder to raise the plate from the surface.
D. After the wash steps, keep the bottom of the Microtiter Filter Plate clean by blotting on paper towels or absorbent pads to prevent any leakage due to capillary action.
E. Keep the vacuum suction on the plate as low as possible. It is recommended to have a vacuum setting that will remove 200 ml of buffer in 4-5 seconds (equivalent to < 100 mmHg).
F. After hydration, all standards and controls must be transferred to polypropylene tubes.
G. The standards prepared by serial dilution must be used within 1 hour of preparation. Discard any unused standards except the 200 ng/ml stock standard which may be stored at £ -20° C for 1 month and at £ -80° C for greater than one month.
H. Any unused Antibody-Immobilized Beads may be stored at 2-8° C for up to one month.
I. During the preparation of the standard curve, make certain to vortex the higher concentration standard well before making the next dilution.
J. We recommend that plates be read immediately after the assay is finished. If the plate cannot be read on the same day as the assay was set-up, seal the plate, cover with aluminum foil, and store at 2-8° C for up to 24 hours. Prior to reading, agitate the plate on the plate shaker for 10 minutes. However, doing so may result in decreased signals and sensitivities for the assay.
K. The titer plate shaker should be set at a speed providing maximum agitation without splashing of liquid outside the wells. For the recommended plate shaker, the setting of 5-7 should be used (approximately 500-800 rpm in a 0.3 cm orbit).
VIII. PREPARATION OF REAGENTS FOR IMMUNOASSAY
A. Preparation of TGF-ß1 Standard
1). Before use, reconstitute the TGF-ß1 Standard with 250 ml Deionized Water to give a 200 ng/ml concentration of standard. Invert the vial several times to mix. Vortex the vial for 10 seconds. Allow vial to sit 5-10 minutes for complete reconstitution. Transfer the standard to an appropriately-labeled polypropylene microfuge tube. This will be used as the 200 ng/ml standard; the unused portions may be stored at £ -20° for up to one month.
2). Preparation of Working Standards
Label five polypropylene microfuge tubes 40, 8, 1.6, 0.32, and 0.064 ng/ml. Add 200 ml of the Assay Buffer to each of the five tubes. Prepare serial dilutions by adding 50 ml of the 200 ng/ml reconstituted standard to the 40 ng/ml tube, mix well and transfer 50 ml of the 40 ng/ml standard to the 8 ng/ml tube, mix well and transfer 50 ml of the 8 ng/ml standard to the 1.60 ng/ml tube, mix well and transfer 50 ml of the 1.60 ng/ml standard to 0.32 ng/ml tube, mix well and transfer 50 ml of the 0.32 ng/ml standard to the 0.064 ng/ml tube and mix well. The 0 ng/ml standard (Background) will be the Assay Buffer.