ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions with Universal Cell Capture Kit Intracellular Compatible User Manual NanoString Technologies, Inc. 530 Fairview Ave North Seattle, Washington 98109 USA Telephone: 206.378.6266 888.358.6266 E-mail: info@nanostring.com Molecules That Count MAN-10032-02 (May 2017)
ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions NanoString Technologies FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Intellectual Property Rights This ncounter Vantage RNA:Protein Immune Cell Signaling Panel for Cell Suspensions and its contents are the property of NanoString Technologies, Inc. ( NanoString ), and is intended for the use of NanoString customers solely in connection with their operation of the ncounter Analysis System. The ncounter Analysis System (including both its software and hardware components) and this User Manual and any other documentation provided to you by NanoString in connection therewith are subject to patents, copyright, trade secret rights, and other intellectual property rights owned by or licensed to NanoString. No part of the software or hardware may be reproduced, transmitted, transcribed, stored in a retrieval system, or translated into other languages without the prior written consent of NanoString. For a list of applicable patents, see www.nanostring.com/company/patents. Limited License Subject to the terms and conditions of sale of the ncounter Analysis System, NanoString grants you a limited, non-exclusive, non-transferable, non-sublicensable, research use only license to use this proprietary nsolver software with the ncounter Analysis System only in accordance with this manual, the manual for the ncounter Analysis System, and other written instructions provided by NanoString. Except as expressly set forth in the terms and conditions, no right or license, whether express, implied, or statutory, is granted by NanoString under any intellectual property right owned by or licensed to NanoString by virtue of the supply of this software or the proprietary ncounter Analysis System. Without limiting the foregoing, no right or license, whether express, implied, or statutory, is granted by NanoString to use the nsolver Analysis Software or ncounter Analysis System with any third party product not supplied or licensed to you by NanoString, or recommended for use by NanoString in a manual or other written instruction provided by NanoString. Trademarks NanoString Technologies, NanoString, the NanoString logo, ncounter, Vantage 3D, nsolver, and Molecules That Count are trademarks or registered trademarks of NanoString Technologies, Inc., in the United States and/or other countries. All other trademarks and/or service marks not owned by NanoString that appear in this document are the property of their respective owners. Copyright 2017 NanoString Technologies, Inc. All rights reserved. 2
NanoString Technologies ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions with Universal Cell Capture Kit Intracellular Compatible Overview ncounter technology can be used to detect a variety of nucleic acids, including mrna, mirna, and DNA. However, other molecules can also be detected using intermediate proxies. NanoString has developed a method for protein analysis using antibodies specific to proteins of interest that have been barcoded with unique synthetic DNA oligonucleotides. Each DNA oligonucleotide is then recognized by a unique Reporter probe that contains a fluorescent barcode. Reporter probes are imaged and counted by the ncounter Analysis System to provide a direct, digital readout of protein expression. This allows for an integrated RNA:Protein workflow. The procedures described in this chapter are compatible with intact cell suspensions from cell lines, PBMCs, and other primary human cells. FFPE and fresh frozen tissue are not compatible with the procedures described in this chapter. Following sample preparation, the RNA and protein components are combined in a single hybridization reaction. Contact NanoString Support (support@nanostring.com) to receive additional assistance with this assay. FIGURE 1 Illustration of the Vantage 3D RNA:Protein Assay workflow. Cells are captured and divided into two fractions which are separately prepared for analysis of RNA or protein expression. The protein sample preparation uses DNA-linked antibodies to recognize proteins of interest. The two analyte preparations are combined for a single Vantage 3D assay. The procedures described are for detection of intracellular proteins from cell suspensions only as indicated by the purple icon in the workflow above. Molecules That Count 3
ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions NanoString Technologies Materials and Reagents TABLE 1. Materials provided in the Vantage 3D RNA:Protein Immune Cell Signaling Panel Kit ncounter Vantage 3D RNA:Protein Reagents Reporter CodeSet Capture ProbeSet Protein Plus Antibody Mix TABLE 2. Materials provided in the Intracellular Compatible Universal Cell Capture Kit Reagent Description Storage Universal Cell Capture Beads Magnetic bead solution 4 C (2 8) C (Do not freeze) Buffer FD Fixation Diluent 4 C (2 8) C Buffer PW Permeabilization and wash 4 C (2 8) C buffer Buffer W Wash buffer 4 C (2 8) C Buffer LH Lysis Buffer Room temperature (15 25) C TABLE 3. Additional materials required (not provided) Material and Reagent Manufacturer Catalog number 96-well plate magnet separator* Thermo Fisher Scientific 12027 Stemcell Technologies 18102 96-well clear polystyrene round bottom plate Corning 351177 Pipettes for 10 1,000 μl* Various Various Manual multi-channel pipette for 200 μl* Rainin L12-200XLS+ 12-strip standard tubes* Bioexpress T-3034-1 15 ml conical tubes* FisherBrand S50712 Hemocytometer* Various Various Trypan Blue* Various Various 1X phosphate buffered saline (PBS; ph 7.4)* Thermo Fisher Scientific 10010-023 Human Trustain FcX** Biolegend 422301 or 422302 Fix buffer concentrate ebioscience 00-5123-43 RNase/DNase free H 2O Thermo Fisher Scientific 4387937 Brefeldin A solution 1000X # Biolegend 420601 * Alternative products can be used if they offer similar function and reliability. ** Only required for samples containing human Fc receptor (e.g., PBMCs). # Only use Brefeldin A solution for fresh cell suspensions. Use with cryopreserved cells may lead to a significant loss in cell viability. 4
NanoString Technologies ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions Protocol A. Advance preparation The following procedure is used for performing 12 reactions. Scale the number of wells according to the number of reactions in your experiment. 1. Prepare fresh 1X Fix Buffer (on day of sample collection) a. Add 700 μl Fix Concentrate to 2.1 ml of Buffer FD. 2. Optional: Treat cells with Brefeldin A a. Add 1000X Brefeldin A to a tissue culture flask containing cells at 1 μl per ml of culture media. b. Incubate at 37 for 4 hours. NOTE: Incubation time may need optimization depending on cell type. NOTE: Extended treatment with Brefeldin A may reduce viability and affect cell yield when performing this assay. Do not treat cryopreserved cells with Brefeldin A. B. Sample collection NOTES: This section is performed in the. 50,000 cells (or 100,000 primary cells such as PBMCs) per sample are recommended for use per reaction. The number of cells can be decreased to a minimum of 20,000 cells (or 50,000 primary cells such as PBMC) if cell numbers are limited but note that using less than the recommended cell number above may result in reduced signal. Using more than the recommended cell number may require increasing the volume of Buffer LH (see Table 4 (Protein Sample Preparation) and Table 5 (RNA Sample Preparation) for details). 1. Determine the concentration of total viable cells in each sample. 2. For each sample, collect the recommended number of cells in 1X PBS containing 2% FBS, or warmed (37 C) cell culture medium in a 1.7 ml microcentrifuge tube. 3. Transfer cell samples to each well in the Top row (A) of the. Add culture media or 1X PBS containing 2% FBS necessary to bring the final volume of each sample well to 200 µl. Molecules That Count 5
ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions NanoString Technologies C. Binding Sample to Universal Cell Capture Beads NOTE: This section is performed in the. 1. Prior to opening the vial of Universal Cell Capture Beads, ensure no beads are on the cap by briefly spinning down, and then thoroughly re-suspend the beads by pipetting. 2. Add 9 µl of Universal Cell Capture Beads to each sample well (A) in the Protein Sample Plate. Use a new pipette tip for each sample well. 3. Mix the samples using a multichannel pipette set to half the sample volume (100 µl). 4. Incubate plate on a flat surface for 30 min at RT. D. RNA sample preparation NOTE: After the sample is divided for RNA and protein detection into the respective plates (Step 1 below), the RNA and protein sample preparation can be performed in parallel. If sequential sample processing is preferred, keep the on ice until ready to proceed with Section F. 1. Mix samples thoroughly by pipetting and transfer 130 µl of each sample from the (Row A) to the corresponding wells in the RNA Sample Plate (Row A). RNA and Protein samples may be processed sequentially or in parallel from this point. 2. Immobilize the bead/cell complexes by placing the RNA Sample Plate on a 96- well plate magnet. Leave plate on the magnet for 5 minutes, undisturbed. 3. Remove and discard the supernatant by either: firmly holding the plate on the magnet and inverting and flicking the plate/magnet only once, followed by blotting of the inverted plate on a fresh paper towel or lab wipe; OR using a single-channel pipette to carefully remove supernatant from each sample well while holding the plate on the magnet. NOTE: Keep the plate in contact with the magnet at all times during this step to avoid sample/bead loss 4. Resuspend cells in 200 µl of Buffer W. RNA Sample Plate 5. Immobilize the bead/cell complexes by placing the RNA Sample Plate on a 96-well plate magnet. Leave plate on the magnet for 5 minutes, undisturbed. 6. Remove and discard the supernatant by either: firmly holding the plate on the magnet and inverting and flicking the plate/magnet only once, followed by blotting of the inverted plate on a fresh paper towel or lab wipe; OR using a single-channel pipette to carefully remove supernatant from each sample well while holding the plate on the magnet. NOTE: Keep the plate in contact with the magnet at all times during this step to avoid sample/bead loss. 7. Without disturbing the bead/cell pellets, use a single channel pipette to carefully remove remaining residual buffer from each sample well. 6
NanoString Technologies ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions 8. Add Buffer LH to each sample well. NOTE: The volume of Buffer LH is dependent upon the initial number of cells in each sample. Refer to Table 4 to determine the volume of Buffer LH to add to each sample well. For example, if starting with 20,000 cells, add 6 µl of Buffer LH to the RNA sample plate. TABLE 4. Buffer LH volume based on initial number of cells Initial Total Cells (cell lines) Initial Total Cells (primary cells) Buffer LH for RNA Lysates 20,000 50,000 6 µl 50,000 100,000 12 µl 9. Pipette thoroughly to lyse cells directly on the beads. Incubate the RNA lysates for 2 3 min at room temperature. NOTE: Avoid creating bubbles during lysis step by setting the pipette to half the volume (e.g., 3 µl in this example of Buffer LH). Failure to do so may result in a loss of sample. NOTE: If the lysate is very viscous, add an additional volume equivalent to Step 8 (e.g., 6 µl in this example) of Buffer LH. 10. Place the RNA Sample Plate on a 96 well plate magnet. Leave plate on the magnet for 5 minutes, undisturbed. Do not discard the supernatant. 11. Without disturbing the bead/cell pellets, carefully collect each RNA lysate/supernatant sample and transfer to a 12-well strip tube using a single channel pipette. 12. Keep the lysates on ice until you are ready to perform Section F: Hybridization. If not using immediately, samples can be stored at -80 C. Molecules That Count 7
ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions NanoString Technologies E. Protein sample preparation NOTES: This section is performed in the. Avoid creating bubbles during wash steps by setting pipettes to half the sample volume. 1. Immobilize the bead/cell complexes in the 70 µl of remaining sample by placing the Protein Sample Plate on a 96-well plate magnet. Leave plate on the magnet for 5 min, undisturbed. 2. Remove and discard the supernatant by either: firmly holding the plate on the magnet and inverting and flicking the plate/magnet only once, followed by brief blotting of the inverted plate on a fresh paper towel or lab wipe, OR using a multichannel pipette to carefully remove supernatant from each sample well while holding the plate on the magnet. NOTE: Keep the plate in contact with the magnet at all times during this step to avoid sample/bead loss. Removing residual liquid after flicking/blotting is not necessary. If buffer is removed by the pipetting method, remove as much of the residual buffer as possible to avoid leaving variable amounts of remaining buffer in the wells. Failure to do so may result in poor quality data. 3. Add 300 µl 1X Buffer PW to Row B of the. 4. Fix the cells by resuspending each of the bead/cell pellets in Row A in 200 µl of 1X Fix Buffer. 5. Incubate at RT for 30 min (there should be buffer and sample in Row A, and buffer only in Row B for pre-blocking). 6. Place the on a 96-well magnet. Leave on the magnet for 5 min, undisturbed. 7. Remove and discard the supernatant from both Row A and Row B. 8. Wash the cells by resuspending each of the bead/cell pellets in Row A with 200 µl Buffer PW and transfer to Row B. 9. Place the on a 96-well magnet. Leave on the magnet for 5 minutes, undisturbed. 10. Remove and discard the supernatant. 11. For primary cells and cell lines expressing human Fc receptors (e.g., cells expressing CD16, CD64, and/or CD32), blocking Fc receptor-mediated antibody binding is necessary to avoid increased background in the assay. If this situation does NOT apply, proceed to Step 12. a. Prepare 1X Fc Receptor Blocking Solution by diluting 65 μl of BioLegend TruStain FcX in 585 μl of Buffer PW (Extra volume is included to account for variation in pipetting). b. Add 50 µl 1X Fc Receptor Blocking Solution to each sample well and thoroughly resuspend beads by pipetting gently. c. Incubate for 10 min at room temperature. d. Add 150 µl Buffer PW to each sample well and proceed to Step 13. 12. Resuspend cells in 200 µl of Buffer PW. 8
NanoString Technologies ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions 13. Incubate at for 30 min at room temperature. 14. Add 10 µl antibody mix (Ab mix) to each sample. Use a new tip for each sample. 15. Thoroughly mix the samples by pipetting gently with pipette set to half the sample volume (100 µl) and incubate for 60 min at RT. 16. Immobilize the bead/cell complex for 5 minutes on the plate magnet. 17. Remove and discard the supernatant from the. 18. Perform a total of 4 washes as follows: a. Remove the plate from the magnet and add 200 µl Buffer PW to each sample well. Mix gently by pipetting. b. Immobilize the bead/cell complex for 5 min on the plate magnet, followed by removal and disposal of the supernatant. c. Repeat Steps 18a b for a second wash. d. Add 200 µl Buffer PW to each sample. Mix gently by pipetting and transfer the samples to the empty row (C). NOTE: Decreased assay sensitivity may occur if this step is not performed. e. Immobilize the bead/cell complex for 5 min on the plate magnet, followed by removal and disposal of the supernatant. f. Repeat Steps 18a b once more. No additional well transfers are necessary. 19. Without disturbing the bead/cell pellets, use a single channel carefully remove remaining residual buffer from each sample well. 20. Add Buffer LH to each sample well. NOTE: The volume of Buffer LH is dependent upon the initial number of cells in each sample. Refer to Table 5 to determine the volume of Buffer LH to add to each sample well. For example, if starting with 20,000 cells for RNA:Protein, add 10 µl of Buffer LH. TABLE 5. Buffer LH volume based on initial number of cells Initial Total Cells (cell lines and primary cells) Buffer LH for Protein Lysates 20,000 10 µl 50,000 25 µl 100,000 50 µl 21. Pipette thoroughly to lyse cells directly on the beads. NOTE: Avoid creating bubbles during lysis step (by setting the pipette to half the volume e.g., 5 µl in this example of Buffer LH). Failure to do so may result in a loss of sample. Molecules That Count 9
ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions NanoString Technologies NOTE: If the lysate is very viscous, add an additional volume equivalent to Step 20 (e.g., 10 µl in this example) of Buffer LH. 22. Transfer cell lysates to a strip tube and incubate for 15 minutes at 95⁰C in a thermocycler with a heated lid at 100⁰C, and then immediately ramp down to 4⁰C or snap cool on ice for a minimum of 2 minutes. 23. Transfer cell lysates back to row (D) of the. 24. Place the on a 96 well plate magnet to immobilize the bead/cell complexes. Leave plate on the magnet for 5 minutes, undisturbed. Do not discard the supernatant. 25. Without disturbing the bead/cell pellets, carefully collect each protein lysate/supernatant sample and transfer to a 12 well strip tube using a single channel pipette. 26. Keep the lysates on ice until you are ready to perform Section F: Hybridization. If not using immediately, samples can be stored at -80 C NOTE: Denaturation of protein lysates is critical for optimal assay performance. It is not necessary to denature the RNA lysates. 10
NanoString Technologies ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions F. Hybridization Total lysate volume (combined RNA and protein) for hybridization should not exceed 5 µl. Mixing should be done by flicking or inverting the tubes. During assay setup, do not vortex or pipette vigorously or shearing of the Reporter Probes may occur. If using a microfuge to spin down tubes, do not spin any faster than 1,000 RCF for more than 30 seconds. Do not pulse to spin because the centrifuge will go to maximum speed and may spin the CodeSet out of solution. IMPORTANT: Check the reagent labels before you begin. For CodeSet assay, see XT CodeSet Vantage RNA:protein hybridization protocol. For TagSet assays, see XT TagSet Vantage RNA:protein hybridization protocol. IMPORTANT: If you are using ncounter Vantage Protein with a Custom CodeSet, run a no-template control to ensure an accurate assessment of background signal. Molecules That Count 11
ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions NanoString Technologies XT CodeSet Vantage 3D RNA:Protein Hybridization IMPORTANT: Pre-heat the thermal cycler to 65 C with a heated lid at 70 C. 1. Remove aliquots of Reporter CodeSet, Protein Plus Reagent and Capture ProbeSet from the freezer and thaw at room temperature. Invert several times to mix well, then spin down reagents. NOTE: Inspect the thawed tubes of Reporter CodeSet and Protein Plus Reagent to make sure no colored precipitate is present. If you see a colored precipitate, heat the entire tube to 75⁰C for 10 minutes and cool at room temperature before using. 2. Create a master mix by adding the following reagents to the tube containing the Reporter CodeSet: 70 µl of Hybridization Buffer 28 µl of Protein Plus Reagent Invert repeatedly to mix, then spin down master mix. NOTE: Do not remove the Reporter CodeSet from this tube. Do not add the Capture ProbeSet to the master mix. 3. Label the hybridization tubes. If using strip tubes, ensure they fit in a microfuge or picofuge (cut the strip in half if necessary). 4. Add 10 µl of master mix to each of the 12 tubes. Use a fresh tip for each pipetting step. 5. Add the volumes of prepared RNA sample (Step 11 of RNA Sample Preparation) and protein sample (Step 25 of Protein Sample Preparation) to each tube as shown in Table 6. TABLE 6. RNA and Protein sample volume input in hybridization* ncounter System RNA lysate (µl) Protein lysate (µl) Nuclease-free water (µl) MAX/FLEX 4 1 0 Sprint 2 0.5 2.5 * Do not exceed 5 µl total lysate volume (RNA and Protein) in hybridization 6. Invert the Capture ProbeSet to mix, then spin down the contents. 7. Add 2 µl of Capture ProbeSet to each tube immediately, then cap tubes and mix the reagents by inverting several times and flicking to ensure complete mixing. 8. Briefly spin down and immediately place the tubes in the pre-heated 65⁰C thermocycler. 9. Incubate hybridization assays for at least 16 hours. Total time at 65⁰C should not exceed 24 hours. 10. Ramp reactions down to 4 C and process the following day. Do not leave the reactions at 4 C for more than 24 hours or increased background may result. NOTE: Selecting a fixed hybridization time followed by a ramp down to 4 C ensures equivalent hybridization time for all assays being directly compared in the same series of experiments. Counts continue to accumulate with time, with total counts typically increasing 5% per hour between 16 and 24 hours. Although a 16-hour incubation is adequate for most purposes, a longer incubation increases sensitivity by increasing counts without significantly increasing background. 12
NanoString Technologies ncounter Vantage 3D RNA:Protein Immune Cell Signaling Panel for Cell Suspensions XT TagSet Vantage RNA:Protein Hybridization IMPORTANT: Pre-heat the thermal cycler to 67 C with a heated lid at 72 C. 1. Remove aliquots of ncounter XT TagSet, Probe A pool, Probe B pool, and Protein Plus from the freezer and thaw on ice. Invert several times to mix well, then spin down reagents. NOTE: Inspect the thawed tube of Protein Plus Reagent to make sure no colored precipitate is present. If you see a colored precipitate, heat the entire tube to 75⁰C for 10 minutes and cool at room temperature before using. 2. Create a 30X Probe A Pool working dilution by adding 22 μl of TE to the 3 μl aliquot of Probe A provided. 3. Create a 30X Probe B Pool working dilution by adding 22 μl of TE to the 3 μl aliquot of Probe B provided. 4. Create a master mix by adding the following reagents to the tube containing TagSet: 70 µl of Hybridization Buffer 28 µl of Protein Plus Reagent 7 μl diluted Probe A 7 μl diluted Probe B 5. Invert repeatedly to mix, then spin down master mix. NOTE: Do not remove the Reporter TagSet from this tube. 6. Label the hybridization tubes. 7. Add 10 μl of master mix to each of the 12 tubes. Use a fresh tip for each pipetting step. 8. Add the volumes of prepared RNA sample (Step 11 of RNA Sample Preparation) and protein sample (Step 26 of Protein Sample Preparation) as shown in Table 25 to each tube. TABLE 7. RNA and Protein sample volume input in hybridization* ncounter System RNA lysate (µl) Protein lysate (µl) Nuclease-free water (µl) MAX/FLEX 4 1 0 Sprint 2 0.5 2.5 *Do not exceed 5 µl total lysate volume (RNA and Protein) in hybridization 9. Cap tubes and mix the reagents by inverting the tubes several times and flicking to ensure complete mixing. 10. Briefly spin down and immediately place the tubes in the pre-heated 67 C thermal cycler. 11. Incubate reactions for at least 16 hours. Maximum hybridization time should not exceed 48 hours. 12. Ramp reactions down to 4 C and process the following day. Do not leave the reactions at 4 C for more than 24 hours or increased background may result. NOTE: Selecting a fixed hybridization time followed by a ramp down to 4 C ensures equivalent hybridization time for all assays being directly compared in the same series of experiments. Counts continue to accumulate with time, with total counts typically increasing 5% per hour between 16 and 24 hours. Although a 16-hour incubation is adequate for most purposes, a longer incubation increases sensitivity by increasing counts without significantly increasing background. Molecules That Count 13