Download nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay

nCounter® Vantage 3D™ RNA:Protein
Immune Cell Profiling Assay for Cell Suspensions
with Universal Cell Capture Kit
Cell Surface Compatible
User Manual
NanoString Technologies, Inc.
530 Fairview Ave North
Seattle, Washington 98109 USA
Telephone: 206.378.6266
888.358.6266
E-mail: [email protected]
Molecules That Count®
MAN-10031-02 September 2016
nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay for Cell Suspensions
User Manual
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
Intellectual Property Rights
This nCounter Vantage 3D RNA:Protein Immune Cell Profiling Assay for Cell Suspensions User Manual 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 the NanoString Patents page.
Limited License
Subject to the terms and conditions of sale of the nCounter Analysis System, NanoString grants you a limited, nonexclusive, 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
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NanoString Technologies, NanoString, nCounter, and Vantage 3D are registered trademarks or 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 manual are the property of their respective owners.
Copyright
© 2016 NanoString Technologies, Inc. All rights reserved.
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NanoString Technologies®, Inc
nCounter® Vantage 3D ™ RNA:Protein Immune Cell Profiling Panel for Cell Suspensions
nCounter Vantage 3D RNA:Protein
Immune Cell Profiling Assay for Cell Suspensions
with Universal Cell Capture Kit
Cell Surface Compatible
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 ([email protected])
to receive additional assistance with this assay.
FIGURE 1. Illustration of the nCounter 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 DNAlinked antibodies to recognize proteins of interest. The two analyte preparations are combined for a single nCounter Vantage
assay. The procedures described are for detection of cell surface proteins only, as indicated by the orange icon in the workflow
above.
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User Manual
Materials and Reagents
TABLE 1. NanoString-provided nCounter Vantage 3D RNA:Protein and Protein Only Immune Cell Profiling Reagents
Kit
Reagents
nCounter Vantage 3D RNA:Protein
Reporter CodeSet
Capture ProbeSet
Protein Plus
Antibody Mix
nCounter Vantage 3D Protein (D)
Protein TagSet
Antibody Mix
nCounter Vantage 3D Protein (R)
Protein Plus
Antibody Mix
TABLE 2. Materials provided in the Cell Surface-Compatible Universal Cell Capture Kit
Reagent
Description
Storage
Universal Cell Capture Beads
Magnetic beads solution
4°C (Do not freeze)
Buffer W
Blocking and Wash buffer
4°C
Buffer LH
Lysis buffer
Room temperature
TABLE 3. Additional materials required (not provided)
Material and Reagent
Manufacturer
Catalog number
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
1.7 mL microcentrifuge tubes*
Various
Various
Hemocytometer*
Various
Various
Trypan Blue*
Various
Various
Human Trustain FcX™**
Biolegend
422301 or 422302
1X phosphate buffered saline (PBS; pH 7.4)*
Thermo Fisher Scientific
10010-023
96-well plate magnet separator*
*Alternative products can be used if they offer similar function and reliability.
**Only required for samples containing human Fc receptor (e.g., PBMCs).
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nCounter® Vantage 3D ™ RNA:Protein Immune Cell Profiling 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.
Protein Sample Plate
1. Pre-block the Protein Sample Plate by adding 300 µL of Buffer W to each of
the 12 wells in two rows of a 96-well round bottom plate.
2. Pre-block RNA Sample Plate by adding 300 µL of Buffer W to each of the
12 wells in one row of a second 96-well round bottom plate.
RNA Sample Plate
3. Block both plates on a flat surface for at least 1 hour at room temperature or
overnight at 4⁰C.
B. Sample Collection
NOTES:
•
This section is performed in the Protein Sample Plate.
•
Use 20,000 cells (or 50,000 primary cells such as PBMCs) from each sample per reaction.
•
Using less than the recommended cell number may result in reduced signal.
•
Using more than the recommended cell number may require increasing the volume of Buffer LH.
See Table 4 (RNA Sample Preparation) and Table 5 (Protein Sample Preparation) for guidelines.
•
Total lysate volume (combined RNA and protein) added to the hybridization (Section F,
Hybridization) should not exceed 5 µL.
•
Perform steps where temperature is not specified at room temperature.
1. Determine the concentration of total viable cells in each sample.
2. For each sample, collect a minimum of 20,000 cells (or 50,000 from primary cell samples) in
Buffer W, 1X PBS containing 2% FBS, or warmed (37°C) cell culture medium in a 1.7 mL
microcentrifuge tube.
3. Remove and discard Buffer W from the Protein Sample Plate by either:
•
inverting and flicking the plate once, followed by blotting of the inverted plate on a fresh
paper towel or lab wipe; OR
•
pipetting out the buffer.
4. Transfer cell samples to each well in the Top row (A) of the Protein Sample
Plate. Add Buffer W if necessary to bring the final volume of each sample
well to 200 µL.
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Protein Sample Plate
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C. Binding RNA:Protein Sample to Universal Cell Capture Beads
NOTE: This section is performed in the Protein Sample Plate.
1. Prior to opening the vial of Universal Cell Capture Beads, ensure no beads are on the cap, 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.
Protein Sample Plate
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 4⁰C.
5. Immobilize the bead/cell complexes by placing the Protein Sample Plate on a 96-well plate
magnet. Leave plate on the magnet for 5 min, 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. 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. The minimum time for magnet pulldown of beads is at least 3 min but 5 min is
recommended. Less than 3 min may result in sample/bead loss and reduced signal in this assay.
7. 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 does NOT apply, proceed to Step 8.
a. Prepare 1X Fc Receptor Blocking Solution by diluting 65 μL of BioLegend TruStain FcX in
585 μL of Buffer W. (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 re-suspend
beads by pipetting gently.
c. Incubate for 10 min at room temperature.
d. Add 150 µL Buffer W to each sample well and proceed directly to Section E: RNA sample
preparation.
8. Add 200 µL Buffer W to each sample well, and thoroughly re-suspend beads by pipetting gently.
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nCounter® Vantage 3D ™ RNA:Protein Immune Cell Profiling Panel for Cell Suspensions
D. RNA Sample Preparation
NOTES:
•
This section is performed in the RNA Sample Plate.
•
After the sample is divided for RNA and protein detection into the respective plates (Step 2), the
RNA and protein sample preparation can be performed in parallel. If sequential sample
processing is preferred, keep the Protein Sample Plate on ice until ready to proceed with
Section E, Protein Sample Preparation.
1. Remove and discard Buffer W used for pre-blocking from the RNA Sample Plate by either:
•
inverting and flicking the plate once, followed by blotting of the inverted plate on a fresh
paper towel or lab wipe; OR
•
pipetting out the buffer.
2. Mix samples from Section C, Binding RNA:Protein Sample to Universal
Cell Capture Beads thoroughly by pipetting gently and transfer 130 µL
of each sample from the Protein Sample Plate (Row A) to the
corresponding wells in the pre-blocked wells of the RNA Sample Plate
(Row A).
Protein Sample Plate
RNA Sample Plate
NOTE: RNA and Protein samples may be processed sequentially or in
parallel from this point.
3. 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.
4. 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 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. The minimum time for magnet pulldown of beads is at least 3 min but 5 min
is recommended. Less than 3 min may result in sample/bead loss and reduced signal in this
assay.
5. Without disturbing the bead/cell pellets, use a single-channel pipette to carefully remove
remaining residual buffer from each sample well.
6. 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 for RNA:Protein, add 6 µL of Buffer LH to the RNA Sample Plate.
7. 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.
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NOTE: If the lysate is very viscous, add an additional volume equivalent to Step 6 (e.g., 6 µL in
this example) of Buffer LH.
TABLE 4. Buffer LH volume based on initial total 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
8. 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.
NOTE: The minimum time for magnet pulldown of beads is at least 3 min but 5 min is
recommended.
9. 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.
10. Keep the lysates on ice until you are ready to perform the Section F, Hybridization. If not using
immediately, samples can be stored at -80°C.
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nCounter® Vantage 3D ™ RNA:Protein Immune Cell Profiling Panel for Cell Suspensions
E. Protein Sample Preparation
NOTES:
•
This section is performed in the Protein Sample Plate.
•
Avoid creating bubbles during wash steps by setting pipettes to half the sample volume.
1. Add 130 µL Buffer W to each sample well (A) in the Protein Sample Plate to
bring the total sample volume to 200 µL.
Protein Sample Plate
2. Add 10 µL antibody mix (Ab mix) to each sample. Use a new tip for each
sample.
3. Thoroughly mix the samples by pipetting gently with pipette set to half the sample volume
(100 µL) and incubate for 30 minutes at 4⁰C.
4. Immobilize the bead/cell complex for 5 minutes on the plate magnet.
5. 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. 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. The minimum time for magnet pulldown of beads is at least 3 min but 5 min is
recommended. Less than 3 min may result in sample/bead loss and reduced signal in this assay.
6. Perform a total of 4 washes as follows:
a. Remove the plate from the magnet and add 200 µL Buffer W to each
sample well. Mix gently by pipetting.
Protein Sample Plate
b. Immobilize the bead/cell complex for 5 min on the plate magnet,
followed by removal and disposal of the supernatant.
c. Repeat Steps 7a–b for a second wash.
d. Add 200 µL Buffer W to each sample. Mix gently by pipetting and
transfer the samples to the empty row (B) of pre-blocked wells.
Protein Sample Plate
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.
Protein Sample Plate
f.
Repeat Steps 7a–b once more. No additional well transfers are
necessary.
7. Without disturbing the bead/cell pellets, use a single-channel pipette to
carefully remove remaining residual buffer from each sample well.
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nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay for Cell Suspensions
User Manual
8. Add Buffer LH to each sample well.
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.
9. Pipette thoroughly to lyse cells directly on the beads. Incubate the protein 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.,
5 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 6 (e.g., 10 µL in
this example) of Buffer LH.
TABLE 5. Buffer LH volume based on initial total cell lines.
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
10. Place Protein Sample Plate 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.
11. 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.
12. Cap the tubes and denature protein lysates only by incubating for 15 min 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.
13. 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.
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nCounter® Vantage 3D ™ RNA:Protein Immune Cell Profiling Panel for Cell Suspensions
F. Hybridization
NOTES:
•
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 assays, 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 notemplate control to ensure an accurate assessment of background signal.
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XT CodeSet Vantage 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 10 of RNA Sample Preparation) and protein
sample (Step 13 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
Protein lysate
Nuclease-free water
MAX/FLEX
4 µL
1 µL
0 µL
Sprint
2 µL
0.5 µL
2.5 µL
*Do not exceed 5 µL total lysate volume (RNA and Protein) in hybridization
6. Invert or flick 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.
NOTE: Minimizing the time between addition of the Capture ProbeSet and incubation at 65⁰C
will increase assay sensitivity.
9. Incubate hybridization assays for at least 16 hours. Total time at 65⁰C should not exceed 24
hours.
10. Hybridizations should be left at 65⁰C until ready for processing. Once removed from the
thermocycler, proceed immediately to post-hybridization processing as described in your
instrument manual. Do not store hybridizations at 4⁰C.
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nCounter® Vantage 3D ™ RNA:Protein Immune Cell Profiling 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.
IMPORTANT: 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
Invert repeatedly to mix, then spin down master mix.
NOTE: Do not remove the Reporter TagSet from this tube.
5. Label the hybridization tubes.
6. Add 10 μL of master mix to each of the 12 tubes. Use a fresh tip for each pipetting step.
7. Add the volumes of prepared RNA sample (Step 10 of RNA Sample Preparation) and protein
sample (Step 13 of Protein Sample Preparation) as shown in Table 7 to each tube.
TABLE 7. RNA and Protein sample volume input in hybridization*
nCounter System
RNA lysate
Protein lysate
Nuclease-free water
MAX/FLEX
4 µL
1 µL
0 µL
Sprint
2 µL
0.5 µL
2.5 µL
*Do not exceed 5 µL total lysate volume (RNA and Protein) in hybridization
8. Cap tubes and mix the reagents by inverting the tubes several times and flicking to ensure
complete mixing.
9. Briefly spin down and immediately place the tubes in the pre-heated 67°C thermal cycler.
10. Incubate reactions for at least 16 hours. Maximum hybridization time should not exceed
48 hours.
11. 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
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nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay for Cell Suspensions
User Manual
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.
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nCounter® Vantage 3D ™ RNA:Protein Immune Cell Profiling Panel for Cell Suspensions
Vantage Protein-only Hybridization Assay
IMPORTANT: Pre-heat the thermal cycler to 65°C with a heated lid at 70°C.
1. Remove an aliquot of Protein TagSet from the freezer and thaw at room temperature. Invert
several times to mix well, then spin down reagents.
NOTE: Inspect the thawed tube of Protein TagSet to make sure no colored precipitate is present.
If you see a colored precipitate, heat the entire tube to 75°C 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 Protein TagSet:
•
70 µL of Hybridization Buffer
•
84 µL of RNase-free water
Invert repeatedly to mix, then spin down master mix.
NOTE: Do not remove the Protein TagSet 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 13 µL of master mix to each of the 12 tubes. Use a fresh tip for each pipetting step.
5. Add the volume of prepared protein sample (Step 13 of Protein Sample Preparation) to each
tube as shown in Table 8.
TABLE 8. Protein sample volume input in hybridization*
nCounter System
MAX/FLEX
Sprint
Protein lysate
Nuclease-free water
1 µL
4 µL
0.5 µL
4.5 µL
*Do not exceed 5 µL total lysate volume (protein) in hybridization
6. Cap tubes and mix the reagents by inverting the tubes several times and flicking to ensure
complete mixing.
7. Briefly spin down and immediately place the tubes in the pre-heated 65°C thermal cycler.
8. Incubate reactions for at least 16 hours. Maximum hybridization time should not exceed
48 hours.
9. 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.
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nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay for Cell Suspensions
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User Manual