ProCartaPoly-Cell Culture.book
Contents
1. Affymetrix Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Affymetrix Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Copyright 2012 Affymetrix Inc All rights reserved Contents Contents Iii Intended Use i a AA 1 Contacting Technical SUPPON sx aa LAND ka k a 2 kk a hala al d4ke Wl son l di 1 About Procarta Immunoassay Kits kk kK KK KK KK KII KK K K KI KIRI KI KIRI KI K KIRI K KI KI KI KI KIRI KK 1 HOW It WOKS oe eo a oboe bee ee oe Gee awa a d aa W OP Oe RD DE ana di i na ERS ORD a 2 Procarta Immunoassay Kit Contents and Storage Conditions 000008 3 Required Equipment and Materials Not Supplied 0 2 00 00 00 ce ee 4 Precautions and Technical Hints eee 4 Sample Preparation sasa aa paha BA kk KK K KIIV ENE KI KK KI KI AA DAGA KIRI KK KI baw Own GA eames 5 Preparing Antigen Standards ua 40 55 wkd dese oon KI KIRI K KI KIR K KIRI KI KI KIR KI KI KIR K KI KI K KI KI KK K 6 Performing the Assay 2 a kk kk kK KK KI K KK KI KI whos KI K K KI KI KI K KI KI KI KI KI KI KI KI KIR KI KI KK ey ee wor 7 Setup of the Luminex Instrument iania ee eee2. 9g 6 g gt NOTE When washing the Filter Plate we recommend using a multi channel pipette and a large plastic reservoir for the wash buffer or a multi channel automatic liquid dispenser Avoid touching the pipette tips to the sides of the wells when adding wash buffer using a multi channel pipette Step 8 Add Premixed Detection Antibodies A Add 25 uL of Detection Antibodies into each well B Gently seal the Filter Plate with a new Plate Seal C Set the Filter Plate onto the Filter Plate Lid and Wrap with aluminum foil Shake at 500 rpm for 30 min at room temperature 8 Procarta Immunoassay User Manual Step Action Step 9 Wash the Filter A Plate Repeat Step 7 Step 10 Add SAPE Vortex the SAPE solution vial for 20 sec Add 50 uL of SAPE solution into each well Gently seal the Filter Plate with a new Plate Seal Set the Filter Plate onto the Filter Plate Lid and wrap with aluminum foil Shake at 500 rpm for 30 min at room temperature Step 11 Wash the Filter A Plate Repeat Step 7 Step 12 Prepare the Add 120 pL of the Reading Buffer into each well Seal the Filter Plate with a new Plate Seal on a Luminex Instrument A Filter Plate for Analysis B C Wrap the Filter with aluminum foil and shake at 500 rpm for 5 min at room temperature D Remove the Plate Seal prior to reading on the Luminex instrument NOTE We recommend reading the assayed sampl
3. beads Biotinylated detection antibody Streptavidin SE SAPE y i do Y X CXA Ig Read signal using a Luminex instrument x A e3 Incubate Incubate Incubate for 60 min for 30 min for 30 min Procarta Immunoassay Kit Contents and Storage Conditions The Procarta Immunoassay Kit contains the following components listed below The kits are available in single 96 well plate or ten 96 well plate formats Refer to the Package Insert for quantities and details of components supplied The kits are shipped with blue ice Shelf life of the kit is 6 months from date of receipt when stored at 2 8 C The kits are also supplied with the following inserts m Packaging Insert Describes the products included in the kit Premixed Standard Insert Lists the lot number and starting antigen values used standard value calculation Bead Analyte Association Insert Lists the bead number and analyte number Component Description Antigen Standards premixed lyophilized 2 vials each lot for a 1 plate kit Recombinant proteins in lyophilized powder Do not reuse discard after use Please note that more than 1 lot of vials may be shipped with each kit Review the Premixed Standard Insert prior to use Detection Antibody premixed Detection antibodies in aqueous buffered solution Antibody Polystyrene Beads Capture antibodies conjugated to microspheres in aqueous buffered solution The Bead ID s are printed
4. 3 pqbhelp affymetrix com Europe 44 1628 552550 techsupport_europe affymetrix com Asia 81 3 6430 430 techsupport_asia affymetrix com About Procarta Immunoassay Kits Procarta Immunoassay Kits are available as m Standard pre mixed panels a By Request user configured panels New custom assay development for analytes not listed on our website Procarta Immunoassay Kits contain all the reagents required to run the assays Please order an appropriate standard diluent buffer for a specific matrix Please contact your local Affymetrix sales representative for new custom assay development for analytes not listed on our website www panomics com 2 Procarta Immunoassay User Manual How it Works Procarta Immunoassays use the xMAP technology multi analyte profiling beads to enable the detection and quantitation of multiple protein targets simultaneously in diverse matrices The xMAP system combines a flow cytometer fluorescent dyed microspheres beads dual laser design and digital signal processing to effectively allow multiplexing of up to 100 unique assays within a single sample The Procarta Immunoassay kits are compatible with all Luminex and Luminex based instruments currently available Capture Detect Label Read on protein captured protein with SAPE Luminex Sample Antibody conjugated Antibody conjugated Antibody conjugated i e beads beads beads ode oy a n S oo Antibody conjugated
5. 8 Pia Pala RESURS 4 ee dd x b SERRE Oho AA 9 Troubleshooting a 9 Setting up and Calibrating the Vacuum Manifold 0 0 0 0 0 0c cee 11 Example Plate L VOL sessi adie akg eas oe KIR k dr Cone hae oe od Boe oe e e ai 14 Blank Plate Layout eee 12 iv Procarta Immunossay User Manual Intended Use This user manual is for a Procarta Immunoassay Kit Polystyrene Beads from Affymetrix to perform quantitative multiplexed immunoassays based on the Luminex technology The procedure is for simultaneous measurements of multiple protein biomarkers in cell culture supernatant samples The assay protocol and reagents supplied are not compatible with other manufacturer s reagents Each 96 well plate kit is configured to allow for the following usage 16 wells for an 8 point standard curve in duplicate 2 wells for blanks and up to 78 wells for samples Procarta Immunoassay kits can be stored for up to 6 months from the date of receipt when stored at recommended temperatures NOTE For the most current version of user documentation go to our website at WWW panomics com Contacting Technical Support For technical support contact the appropriate resource provided below based on your geographical location For an updated list of FAQs and product support literature visit our website at WwWw panomics com Location Contact Information North America 1 877 726 6642 option 1 then option
6. on the inside flap of the Kit Box and also listed in the Bead Analyte Associate Insert Streptavidin PE SAPE Streptavidin conjugated R phycoerythrin in aqueous buffered solution 10X Wash Buffer Concentrated aqueous buffered solution Reading Buffer Aqueous buffered solution PCR 8 Tube Strip 0 2 mL polypropylene PCR 8 tube strip Filter Plate and Lid Plate Seals 1 Contains sodium azide See WARNING below 96 well Filter plate used with the vacuum manifold Adhesive backed foil plate sealer A WARNING All chemicals should be considered potentially hazardous We recommend that this product and its components be handled by those trained in laboratory techniques and be used according to the principles of good laboratory practice a WARNING This kit contains small quantities of sodium azide Sodium azide is highly toxic and reactive in the pure form At this product s concentration though not classified as hazardous build up of sodium azide may react with lead and copper plumbing to form highly reactive explosive metal azide Dispose of the product in accordance with all State and local regulations 4 Procarta Immunoassay User Manual Required Equipment and Materials Not Supplied Required Equipment Material Source Part Number Vacuum Manifold Millipore Microplate shaker Labline 4625 or equivalent must have 3 mm orbit with ability to maintain 500 rpm Luminex or Lumin
7. 12 12 20 20 28 28 36 3 6 6 13 13 21 21 29 29 37 37 7 7 14 14 22 22 30 30 38 38 Blank 15 15 23 23 31 31 39 39 Blank Plate Layout 1 2 3 7 10 11 12 A B C D E F G H
8. KGS Panomics Atfymetrix Solutions User Manual Procarta Immunoassays Using Polystyrene Beads For cell culture supernatant samples P N 10482 CC Rev A 020912 ii Procarta Immunossay User Manual For research use only Not for use in diagnostic procedures Trademarks a Affymetrix AW and Procarta are registered trademarks of Affymetrix Inc Luminex xMAP and xPonent are registered trademarks of the Luminex Corporation All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Citing Procarta Immunoassay in Publications When describing a procedure for publication using this product please refer to it as the Procarta Immunoassay from Affymetrix Disclaimer
9. cubate on ice for 5 10 min Instructions for assay panels with more than one standard lots in the kit Centrifuge all the antigen standard vials with different lot numbers at 2000 x g for 10 sec Add 250 uL of fresh cell culture media into one of the vials Incubate the vial on ice for 5 10 min Vortex gently for 30 sec Transfer the entire content into the second vial with a different lot number Incubate on ice for 5 10 min Vortex gently for 30 sec CON DU BB U N s If more than 2 lots of antigen standards are in the kit repeat steps 5 7 until all the vials with different lot numbers are reconstituted NOTE For panels with liquid antigens follow the instructions on the Package Insert Step 2 Prepare 4 Fold Serial Dilution g IG mm m Prepare a 4 fold serial dilution of the reconstituted standard s using the PCR 8 tube strip provided Add 200 uL of the reconstituted antigen standards into the first tube of the strip tube and label as Standard 1 Std 1 Add 150 uL fresh cell culture media into Tubes 2 8 Using a P 200 pipette transfer 50 uL of the reconstituted antigen standards from Tube 1 into Tube 2 Mix by pipetting up and down for a total of 10 times After changing the pipette tip transfer 50 uL of the mixed standards from Tube 2 into Tube 3 Mix by pipette up and down 10 times Repeat Actions D to G for the rest of the tubes to prepare Std 4 8 Transfer 200 uL 50uL 50uL 50
10. e may have a different Std 1 concentration A 4 fold dilution should be applied to each subsequent standard Standard 2 8 For example if the starting concentration was 20 000 then a 4 fold dilution for Std 1 8 would be 20 000 5000 1250 312 78 19 5 4 8 and 1 2 pg ml NOTE If there is a malfunction of the Luminex instrument or software during the run the Filter can be re read Remove the Filter Plate from the instrument and vacuum filter the plate Resuspend the beads in 120 uL of Reading Buffer seal with a new Plate Seal and shake at 500 rpm for 5 min at room temperature The assayed samples may take longer to read since there will be less beads in the wells Analyzing Results The concentration of the samples can be calculated by plotting the expected concentration of the standards against the MFI generated by each standard A 4PL or 5PL algorithm is recommended for the best curve fit Analyze the assayed samples according to the operation manual for the Luminex or Luminex based instrument Troubleshooting Observation Low Flow Rate Probable Cause Partial Blockage of the flow cell Recommend Solution Remove the Filter plate and perform a wash and rinse cycle Instrument needle is partially clogged Replace or clean needle according to the manufacturer s recommendations High CVs Samples and antigen standards not stored on ice Prepare the samples and standards on ice before setting
11. er Plate to form a tight seal C Adjust the pressure so that it takes 4 6 seconds to evacuate 150 uL of wash buffer from the wells If the vacuum is too high beads can get trapped or pulled through the filter D Turn off vacuum as soon as the solution filters through the wells and remove the plate from the manifold Step 3 Operating the Manifold For all filtration steps turn on the vacuum pump place the Filter Plate onto the vacuum manifold and then filter the solution Avoid splashing and cross contamination of wells during all wash steps NOTE Do not allow the Filter Plates to air dry following washes and immediately add the next component after each filtration step We recommend performing all the wash steps next to the manifold to minimize the amount of time that the beads are exposed to air s Step 4 Following the last wash in each series blot the bottom of the Filter Plate thoroughly with a paper towel to remove traces of 1X Wash Buffer Avoid touching the bottom of the Filter Plate with your fingers or to the bench during manipulations Immediately move to the next step to ensure that the beads are in the appropriate buffered solution 12 Procarta Immunoassay User Manual Example Plate Layout Standards Samples 1 1 8 8 16 16 24 24 33 32 3 2 9 9 17 17 25 95 CE 33 3 3 10 10 18 18 26 26 34 34 4 4 11 11 19 19 27 27 35 35 5
12. es on the Luminex instrument immediately However the Filter Plate can be wrapped with aluminum foil and stored for up to 48 hours at 4 C before proceeding After storage shake at 500 rpm for 5 min at room temperature prior to reading Delay in reading the assayed samples may result in decreased sensitivities for some assays Setup of the Luminex Instrument Software Sample Size DD Gate Timeout Bead Event Bead Region Luminex 100 100 uL 7 500 15 000 45 sec 50 100 IS100 v 2 318 Bioplex Bio Rad 100 uL 4 335 10 000 45 sec 50 100 Miraibio Hitachi 100 uL 2 000 15 000 45 sec 50 100 Starstation ACS 100 uL 4 335 10 000 45 sec 50 100 If you are running assays on your Luminex instrument that uses both Plates for Magnetic Beads and Filter Plates for Polystyrene Beads verify the probe height for each plate type before reading Failure to adjust the probe height can cause damage to the instrument The Luminex system allows for calibration of Low and High RP1 target values We recommend RP1 Low target value settings for Procarta Immunoassays Please refer to the label inside of the Kit Box or the Bead Analyte Association Insert for bead region analyte associations when entering the information into the Luminex acquisition software xPonent Bio Plex MasterPlex StarStation Please also refer to the Premixed Antigen Standard Insert when assigning the Standard 1 Std 1 concentration into the analysis software Each analyt
13. ette and reagent reservoirs whenever possible to achieve optimal assay precision Store the detection antibody antibody beads Streptavidin PE samples and reconstituted standards on ice before adding to the Filter Plate For frozen samples thaw completely on ice and mix well prior to running the assay Seal all unused wells with an enclosed Plate Seal to ensure proper vacuum pressure When sealing the Filter plate with a Plate Seal gently press your finger over the Plate Seal Too much pressure can force the fluid through the filter plate To avoid Filter Plate leakages do not seal Filter Plates using a rubber roller or equivalent as they apply significant pressure resulting in leakage Sample Preparation Cell culture supernatant samples can be obtained from adherent and non adherent cells A total volume of 50 uL well of cell culture supernatant is needed and a minimum of 2 replicates is recommended Some samples may contain high concentrations of the analytes Dilution of the samples may be needed if the analyte concentration is above the assay upper limit of quantitation Serial dilution of the samples may need to be prepared to determine the appropriate dilution factor for accurately measuring the analytes of interest Use the same fresh culture medium used for culturing the experimental cells to prepare dilutions of the samples If the sample contains cells centrifuge the sample at 300 x g for 5 min at 2 8 C Collect the
14. ex based instrument MiraiBio Bio Rad or other Luminex instrument providers Vortex mixer Major laboratory supplier MLS Adjustable single and multi MLS channel precision pipettes for dispensing 1 20 uL 20 200 uL and 200 1000 uL Reagent reservoirs 25 mL and Vist Labs 3054 1002 or equivalent 100 mL capacity Corning Costar CLS 4873 or equivalent Double distilled dd water MLS H 0 Microcentrifuge MLS Precautions and Technical Hints a Thoroughly read this user manual and product insert that is included with the assay kit The product insert may contain specific instructions for proper use of the By Request or custom panels Before starting the assay turn on the Luminex machine and initiate the startup protocol It takes 30 min for the lasers to warm up Make sure the Luminex machine is calibrated according to the manufacturer s instructions Some samples may contain high analyte concentrations and require sample dilution for accurate quantitation Use the cell culture media used to prepare the cells a When working with samples and standards change the pipette tips after every transfer and avoid creating bubbles when pipetting a During the incubation steps cover the 96 well plate with aluminum foil to minimize exposure of the beads to light Be careful not to invert the Filter Plate during the assay or allow contents from one well to mix with another well a Use a multi channel pip
15. instrument Instrument needle is partially clogged Replace or clean needle according to the manufacturer s recommendations Beads settle on the bottom of the well Confirm that the plate shaker is set to 500 rpm and shaking for at least 5 min before reading Air bubble in the sample loop Refer to the Luminex manual for proper removal of the air bubble Low signal or sensitivity Standards not reconstituted and diluted correctly Expired reagents were used Prepare fresh antigen standards following the instructions in the Preparing Antigen Standards Section Reagents are good for 6 months from the date of receipt Do not use expired reagents Suboptimal assay conditions Follow the recommended incubation times and temperature Shake the Filter Plate during all incubations except during optional overnight incubation step Step 7C Poor accuracy Did not use the appropriate assay diluents Use the same sample type specific standard and assay buffers for standard and sample preparations Samples and antigen standards were not stored on ice Prepare and store the samples and standards on ice before setting up the assays 11 Setting up and Calibrating the Vacuum Manifold Step Action Step 1 Set Up Vacuum Manifold Step 2 Calibrate Pressure A Place the Filter Plate on top of the manifold and Turn on the vacuum B Press down on all 4 corners of the Filt
16. supernatant Use immediately or aliquot and store at 80 C The TGF p assay requires a special sample preparation procedure The TGF assay can only detect the active form of TGF f The samples must be acid treated and then neutralized to convert the complexed form of TGF to its active form The assay should be processed as a single plex assay since the sample must be acid treated The TGF B sample preparation protocol can be found on our website WWwW panomics com 6 Procarta Immunoassay User Manual Preparing Antigen Standards This section provides instructions on how to make a 4 fold 8 point standard curve for the assay panel The antigen standards should be prepared after sample preparation is completed The serially diluted antigen standards should be added to the assay plate at the same time the samples are added Each1 plate kit is shipped with two vials of identical antigen standards from the same lot In some cases an additional set s of standards from a different lot may be included in the kit Please refer to the Premixed Antigen Standard Insert when assigning the Standard 1 Standard 8 antigen values for each analyte Step Action Step 1 Reconstitute Lyophilized Antigen Standards A Instructions for assay panels with only 1 standard lot in the kit 1 Centrifuge the antigen standard vial at 2000 x g for 10 sec 2 Add 250 uL of fresh cell culture media into the vial 3 Vortex gently for 30 sec 4 In
17. tyrene B Add 50 pL of Antibody Beads to each well Beads C Remove buffer with vacuum filtration Step 4 Wash Antibody A Add 150 uL of 1X Wash Buffer into each well and remove by vacuum filtration Beads B Blot the bottom of the Filter Plate thoroughly with paper towels to remove residual buffer Step 5 Add Standards and Samples Add 50 uL of standards or 50 uL of samples as marked on the plate layout sheet into each well For blanks add 50 uL of cell culture media Step 6 Incubate the Filter Plate A Gently seal the Filter Plate gently using a Plate Seal provided place the Filter Plate onto the Filter Plate Lid and completely wrap with aluminum foil B Room temperature incubation 1 Shake the 96 Well Plate at 500 rpm for 60 min at room temperature 2 Proceed to step 7 C Alternatively the 96 Well Plate can be incubated overnight 1 Shake the Filter Plate at room temperature for 30 min 2 Transfer the plate to 4 C and store on a level surface 3 After incubation remove the Filter Plate from 4 C and shake for 30 min at room temperature 4 Proceed to step 7 Step 7 Wash the Filter Plate Carefully remove the Plate Seal to avoid splashing the plate contents Remove solution with vacuum filtration Add 150 uL of 1X Wash Buffer into each well then immediately remove with vacuum filtration Wash the plate three times and after the 3 4 wash thoroughly blot the bottom of the Filter Plate with paper towels
18. uL 50uL 50puL 50uL 50 uL Antigen Standard Vial Std1 Std2 Std3 Std4 Std5 Std6 Std7 Std 8 Performing the Assay Step Action Step 1 Prepare 1X wash buffer Bring the 10X Wash Buffer to room temperature and vortex for 15 seconds Mix 20 mL of the 10X Wash Buffer with 180 mL ddH20 NOTE 1X wash Buffer can be stored at 2 8 C for up to 6 months Bring the buffer to room temperature prior to use Step 2 Prepare the Filter Plate A Mark the standard sample and blank wells For your convenience a blank layout is provided in the Plate Layout section B Seal the un used wells of the plate with a Plate Seal prior to starting the assay Keep the un used wells of the plate sealed throughout the whole assay procedure Place the Filter plate on top of the inverted Filter Plate Lid The plate should be sitting on the hollow end of the lid and not touching any of the surfaces except for the borders of the lid This will prevent the bottom of the Filter Plate from against any surface which can lead to leakage C Pre wet the Filter Plate by adding 150 uL of Reading Buffer into each well D Incubate for 5 minutes at room temperature E Remove the Reading Buffer with vacuum filtration Reading Buffer should clear wells within 8 10 seconds See Setting Up and Calibrating the Manifold at the end of this manual Step 3 Add the A Vortex the premixed Antibody Beads for 30 seconds at room temperature Antibody Polys
19. up the assay Contamination from re using the Plate Seal Use a new Plate Seal for each incubation step Contamination from contents from adjacent wells Poor pipetting techniques Avoid splashing the Wash Buffer during wash steps into adjacent wells Use appropriate pipetting techniques Use new pipette tips for each well during sample and standard addition Avoid touching pipette tips to sides of the wells when adding wash buffer 10 Procarta Immunoassay User Manual Observation Probable Cause Recommend Solution Low bead count Probe height is incorrect Refer to the Luminex manual for proper adjustment of the needle height Reading buffer volume added in the last step to resuspend the beads is too low Add 120 uL Reading Buffer into each well and shake at 500 rpm for 5 min at room temperature to resuspend the beads prior to reading on the Luminex instrument Make sure sample size is set at 100 uL in the acquisition protocol High bead aggregation Vortex the bead suspension well before using in the assay and ensure that the beads are properly mixed during the incubation steps Dyes contained in the beads are photo bleached from overexposure to light Store bead solution in the dark and protect the Filter Plate from light by wrapping the Filter Plate with aluminum foil Partial blockage of the flow cell Remove the Filter Plate and perform a wash and rinse to the
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