CG000529 | Rev C

DEMONSTRATED PROTOCOL

Cell Surface & Intracellular Protein Labeling for
Chromium Fixed RNA Profiling
Introduction
Chromium Fixed RNA Profiling (Gene Expression Flex) with Feature Barcode technology offers comprehensive,
scalable solutions to measure gene and protein expression in formaldehyde fixed samples. To measure protein
expression, cell surface and intracellular proteins are labeled using an antibody conjugated to a Feature Barcode
oligonucleotide, followed by a fixation with formaldehyde to ensure 1) the cells are properly fixed andpermeabilized for probe hybridization and 2) the antibodies firmly bind to the cells, preventing their loss duringprobe hybridization.
This document outlines cell surface and intracellular protein labeling protocols for use with Chromium Fixed
RNA Profiling. General guidance on antibody-oligonucleotide conjugation, evaluating antibodies with flow
cytometry, and best practices when staining cells is also provided here.
Both the cell surface and intracellular protein labeling protocols described in this document require a Fc
receptor blocking step, after which the appropriate labeling protocol should be followed depending on the typeof antibodies used. The Intracellular Protein Labeling Protocol, which includes a brief fixation before staining toallow cell permeabilization, can also be used with certain fixation compatible cell surface protein antibodies.

Fc BlockChoose one of the following

Cell Surface Protein Labeling• Label viable cells with antibodies
targeting cell surface antigens

Intracellular Protein Labeling• Brief fixation required• Label fixed cells with antibodies
targeting intracellular antigens orboth intracellular & cell surface
antigens**Not all cell surface antibodies arecompatible with brief fixation required
before staining; consult manufacturer's
guidance on compatibility

Cell Surface Protein Labeling• Label viable cells with antibodies
targeting cell surface antigens
thenIntracellular Protein Labeling• Brief fixation required• Label fixed cells with antibodies
targeting intracellular antigens or
intracellular & cell surface antigens*

Fix Cells (Consult CG000478 for complete protocol)
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Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

Additional GuidancePreread and have available Fixation of Cells &Nuclei for Chromium Fixed RNA Profiling
Demonstrated Protocol (CG000478) before starting
the protocols provided in this document. Consult
the Cell Preparation Guide Handbook (CG00053)
for Tips & Best Practices on handling and counting
cells. Consult Cell Thawing Protocols for Single Cell
Assays (CG000447) for guidance on thawing
dissociated tumor cells.
Consult Chromium Fixed RNA Profiling - Protocol
Planner (CG000528) for details on workflow
overview, document resources, and guidance on
selecting the appropriate sample preparation andlibrary construction protocols for different
Chromium Fixed RNA workflows.
Cells carry potentially hazardous pathogens.
Follow material supplier recommendations andlocal laboratory procedures and regulations forthe safe handling, storage, and disposal of
biological materials.

Specific Reagents & Consumables
For Cell Surface Protein Labeling
Vendor

Item

Part Number

BioLegend

Human TruStain FcX (Fc
Receptor Blocking Solution)

422301

TruStain FcX PLUS (antimouse CD16/32) Antibody

156604True-Stain Monocyte Blocker

426101

TotalSeqTM-C Human Universal 399905
Cocktail, V1.0
TotalSeq -B Human Universal 399904
Cocktail, V1.0
TM

Choose appropriate antibodies
based on the Chromium
Fixed RNA Profiling workflow.
See TotalSeqTM AntibodyOligonucleotide Conjugates
Cell Staining Buffer

CG000529 | Rev C

ThermoFisherScientific

UltraPure Bovine Serum
Albumin (BSA, 50 mg/ml)

AM2616

Fetal Bovine Serum, qualified,heat inactivated16140071

Millipore
Sigma

Bovine Serum Albumin In
DPBS (10%) Alternative to
Thermo Fisher

A1595

Miltenyi
Biotec

MACS BSA Stock Solution
Alternative to Thermo Fisher

130-091-376

Corning

Phosphate-Buffered Saline,
1X without Calcium andMagnesium

21-040-CV

VWR

Fetal Bovine Serum (FBS)
Alternative to Thermo Fisher

97068-085

For Cell Counting
ThermoFisherScientific

Nexcelom
Bioscience

Countess II FL Automated Cell
Counter

AMQAF1000

Countess 3 FL Automated Cell
Counter

A49866Countess Cell Counting
Chamber Slides

C10228Trypan Blue Stain (0.4%)

T10282

Celleca MX High-throughput
Automated Cell Counter

MX-112-0127

ViaStain AOPI Staining
Solution

CS2-0106-5mL

Cellometer K2 Bundle w/
Matrix Software

CMT-K2MX-150

VS Cellometer AOPI Staining
Solution

CS2-010625ML

PD100 Counting Chambers 1
case

CHT4PD100-003

This list may not include some standard laboratory equipment.
TotalSeqTM Antibody-Oligonucleotide Conjugates
Chromium Fixed RNA
Profiling Workflows

TotalSeqTM AntibodyOligonucleotide Conjugates

Gene & Protein
Expression using
Barcode Oligo
Capture – Singleplex &Multiplex Workflows

TotalSeqTM-C antibodyoligonucleotide conjugates

Gene & Protein
TotalSeqTM-B antibodyExpression – Singleplex oligonucleotide conjugates
Workflow
420201

Choose appropriate TotalSeqTM antibody-oligonucleotide
conjugates based on the Chromium Fixed RNA Profilingworkflow.
10xgenomics.com

2

Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

For Intracellular Protein Labeling
Vendor10x
Genomics

Item
Chromium Next GEM Single
Cell Fixed RNA Sample
Preparation Kit*

Part Number
1000414

BioLegend

Human TruStain FcX (Fc
Receptor Blocking Solution)

422301

TruStain FcX PLUS (antimouse CD16/32) Antibody

156604True-Stain Monocyte Blocker

426101

TotalSeqTM intracellular
conjugates-

Proteintech MultiPro Human Fixed Cell
Genomics
Immune Profiling Antibody
cocktail

G900004

Corning

Phosphate-Buffered Saline,
1X without Calcium andMagnesium

21-040-CV

ThermoFisherScientificTween 20

Additional Materials
Eppendorf

DNA LoBind Tubes 2.0 ml

022431048

1.5mL Protein LoBind tubes

022431081

1.5mL DNA LoBind tubes

022431021

ThermoMixer C

5382000023

VWR

Vortex Mixer

10153-838

Corning

Phosphate-Buffered Saline,
1X without Calcium andMagnesium

21-040-CV

New
England
Biolabs

T4 Gene 32 Protein**

M0300S/
M0300LFor Cell Counting
Countess II FL Automated Cell
Counter

AMQAF1000

28320

Countess Cell Counting
Chamber Slides

C10228NP-40 Surfact-Amps
Detergent Solution (10%)

85124

Trypan Blue Stain (0.4%)

T10282Nonidet P40 Substitute,
Ultrapure, Thermo Scientific
Chemicals
Alternative to NP-40; consult
manufacturer's instructions for
preparing 10% stock solution
Formaldehyde (37% by
Weight/Molecular Biology),
Fisher BioReagents
Nuclease-free Water (notDEPC-Treated)

J19628.AP

Celleca MX High-throughput
Automated Cell Counter

MX-112-0127

ViaStain AOPI Staining
Solution

CS2-0106-5mLAlbumin, Bovine Serum,
Fraction V, Fatty Acid-Free,
Nuclease- and Protease-Free

126609Dextran Sulfate Sodium Salt
8 KDa

RES2029D-A7

UltraPure Salmon Sperm DNA
Solution

15-632-011

Phosphate-Buffered Saline(10X) pH 7.4, RNase-free

AM9625

AvantorPBS 1X

K812-500ML

Roche

Protector RNase inhibitor

3335399001

Millipore
Sigma

Invitrogen

ThermoFisherScientific

Nexcelom**Optional: Only required if adding to the antibody pool +buffer to reduce background signal.
BP531-25

AM9937*Chromium Next GEM Single Cell FixedRNA Sample Preparation Kit, 16 rxns PN1000414
Chromium
Single Cell Fixed RNA Sample Preparation
Kit
16 rxns, PN-1000414# PN
Store at -20°C
Conc. Fix & Perm Buffer
Conc. Quench Buffer
Enhancer

3
6120005172000516
2000482

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3Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Antibody-Oligonucleotide Conjugates• The Cell Surface Protein Labeling protocol described in this document was optimized using
preconjugated TotalSeqTM-C or TotalSeqTM-B antibodies from BioLegend. Choose appropriate antibodyoligonucleotide conjugates based on the Chromium Fixed RNA Profiling workflow (see the table below).
• The Intracellular Protein Labeling protocol was optimized using preconjugated TotalSeqTM intracellular
conjugates from Biolegend & MultiPro Fixed Cell Immune Profiling Antibody Cocktail, a pool of cellsurface, intracellular, & isotype control antibodies, from Proteintech Genomics.
• Antibodies conjugated to appropriate Feature Barcode oligonucleotide from other vendors can also be
used. See the table below for antibody-oligonucleotide conjugate capture by protocol-specific Gel Beadprimers.
• Both the labeling protocols are also compatible with custom conjugated antibody panels.

Antibody-Oligonucleotide Conjugate Capture
Chromium RNA Fixed RNA Profiling Workflows

Antibody-Oligonucleotide Conjugates

Gene & Protein Expression using Barcode Oligo
Capture* - Singleplex & Multiplex Workflows/5AmMC12/CGGAGATGTGTATAAGAGACAG-N10-N15-N9-CCCATATAAGAAA

Consult the following User Guides for more
information:• Chromium Fixed RNA Profiling Reagent Kits
for Multiplexed Samples with Feature Barcode
technology for Protein using Barcode Oligo
Capture User Guide (CG000673).

Nextera partial Read 2
Feature Barcode (15 nt) Capture Sequence
TotalSeqTM-C antibody-oligonucleotide conjugates/5AmMC12/CGGAGATGTGTATAAGAGACAG-N15-CCCATATAAGAAA

Nextera partial Read 2 Feature Barcode (15 nt) Capture Sequence
MultiPro Fixed Cell Immune Profiling Antibody Cocktail
Gel
Bead

• Chromium Fixed RNA Profiling Reagent
Kits for Singleplexed Samples with Feature
Barcode technology for Protein using Barcode
Oligo Capture User Guide (CG000674).
Gene & Protein Expression - Singleplex
Workflow
Consult the following User Guide for more
information:• Chromium Fixed RNA Profiling Reagent
Kits for Singleplexed Samples with Feature
Barcode technology for Protein User Guide(CG000477).

-TTGCTAGGACCG-3’

TruSeqRead 1

10x UMI
BarcodePartial Capture Sequence 1 (pCS1)/5AmMC12/GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-N10-N15-N9-GCTTTAAGGCCGGTCCTAGCAA

TruSeq Read 2
Feature Barcode (15 nt)
Capture Sequence 1
TotalSeqTM-B antibody-oligonucleotide conjugates
Gel
Bead

-TTGCTAGGACCG-3’

TruSeqRead 1

10x UMI Partial Capture Sequence 1 (pCS1)
Barcode3’-AACGATCCTGGCCGGAATTTCG

Capture Sequence 1

FeatureBarcode

TruSeq Read 2*Barcode Oligo Capture can be achieved by using either TotalseqTM-C or MultiPro Fixed Cell Immune Profiling Antibody
Cocktail. If using both the antibodies in a single experiment, an alternative sequencing configuration will be required.
Consult 10x Genomics support website for details.
Conjugation Guidance• Follow manufacturer's instructions (Antibody Conjugation Kit from Abcam and oYo-Link Antibody
Labeling Reagents from AlphaThera) for antibody-oligonucleotide conjugation.
• Antibody conjugation services from Abcam and AlphaThera can also be utilized.
• Consult Cell Surface Protein Labeling for Single Cell RNA Sequencing Protocols with Feature Barcode
technology Demonstrated Protocol (CG000149) for guidance on antibody-oligonucleotide conjugationand verification of conjugation.
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4Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

Fc Receptor Blocking

CG000529 | Rev C

If using >1 x 106cells, scale up the buffer volumes
accordingly.

Both cell surface and intracellular labelingprotocols require a Fc receptor blocking step. After
this step, follow the appropriate labeling protocol
based on the type of antibodies used.

f.

Buffers – Preparation

Add 5 μl Human TruStain FcX or 2 μl
TrueStain FcX PLUS (anti-mouse CD16/32
Antibody). Gently pipette mix.
OPTIONAL: If performing cell surface proteinlabeling, 5 μl True-Stain Monocyte Blocker canalso be added at this step in addition to TruStain
FcX to reduce staining background.

• Chilled (4°C): PBS + 1% BSA• For samples containing <70% viable cells, chilled(4°C) PBS + 10% FBS can be used.
a. Resuspend cells in PBS + 1% BSA.

Consult manufacturer's instruction for therecommended volume of Fc receptor blocking
solutions.

For samples containing <70% viable cells, chilled(4°C) PBS + 10% FBS can be used.
b. Transfer ≤1 x 106 cells to a new 1.5-ml
microcentrifuge tube.

g. Incubate for 10 min at 4°C.
h. Immediately proceed to the appropriate step:c. Centrifuge cells at 400 rcf for 5 min (PBMCs)
at 4°C. Use of a swinging-bucket rotor isrecommended for higher cell recovery.
Centrifugation speed and time depend upon
the sample type. Larger or fragile cell types
may require slower centrifugation speeds.

• Cell surface protein detection only: proceed toCell Surface Protein Labeling• Intracellular protein detection only and/or using cell surface antibodies that arecompatible with brief fixation: proceed to
Intracellular Protein Labelingd. Remove the supernatant without disturbing thepellet.
e. Resuspend pellet in 50 μl chilled PBS + 1% BSAor chilled PBS + 10% FBS (for <70% viable cells).

• Both cell surface protein and intracellular
protein detection: proceed to Cell Surface
Protein Labeling followed by IntracellularProtein Labeling

Block Fc receptor

Resuspend in
400 rcf,
Remove
PBS + 1% BSA (50 μl)
Cells in 5 min, 4°C supernatant Add Human TruStain
PBS +FcX (5 μl) or TruStain
1% BSA*FcX PLUS (2 μl)
Gently pipette mix

Incubate for10 min (4°C)

ChooseprotocolCell Surface Protein
Labelingor(pg 6-11)

Intracellular Protein Labeling(pg or12-17)

Both Protein Detection(perform Cell Surface
Protein labeling first, thenIntracellular Protein Labeling)*For samples containing <70% viable cells, PBS + 10% FBS can be used.
10xgenomics.com

5Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Cell Surface Protein
Labeling

Cell Surface Protein Labeling Protocol
Cell Surface Protein Labeling Protocol Overview

Label Cells

Wash Cells(no wash, 1-wash, or
2-wash options)This protocol was optimized using TotalSeqTM-C
and TotalSeqTM-B antibody-oligonucleotide
conjugates from BioLegend. The labeled cells canbe enriched by flow sorting (see Appendix).

Use distinct and compatible antibody clones forflow sorting and cell surface protein labeling.
Optimize the working concentration of eachantibody used.
Using the appropriate wash option is critical for
obtaining high-quality data and minimizing
background signal. Choose a washing protocol
based on the guidance outlined in the protocol.
See Appendix for supplemental data on the
different wash options.

Buffers – Preparation
For Labeling Cells• Chilled (4°C): PBS + 1% BSA• For samples containing <70% viable cells, chilled(4°C) PBS + 10% FBS can be used.

Fix Cells** Consult Fixation of Cells & Nuclei
for Chromium Fixed RNA Profiling
Demonstrated Protocol (CG000478).

Prepare Antibody Mix Supernatant
Add the appropriate/manufacturer's recommended
amount of antibody-oligonucleotide conjugates to a
1.5-ml Protein LoBind tube.
• If using lyophilized antibody panel/cocktails,
rehydrate the lyophilized panel in therecommended volume of Cell Staining Buffer
as directed from BioLegend. Follow the
manufacturer's instructions for use for celllabeling. Perform cell wash steps as described inthis Demonstrated Protocol.
• Centrifuge the mix at 14,000 rcf for 10 min at 4°C.
• Transfer the supernatant (containing Antibody
Mix) to a new protein Lobind tube. Leave residual
volume so the antibody aggregates are notcarried over. Maintain at 4°C.

Prepare Flow Sorting Antibody Pool
• Add the appropriate/manufacturer's
recommended amount of fluorophore-conjugatedantibodies to a 1.5-ml microcentrifuge tube on
ice.
• Gently pipette mix and maintain at 4°C. Avoid
light exposure.

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6Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Cell Surface Protein
Labeling

Tips & Best Practices
Cell Viability

Sample Washing• Determine sample viability before starting the cellsurface protein labeling protocol.

• This protocol provides three wash options after
incubation with the antibody-oligonucleotide
conjugate. See Cell Surface Protein Labeling:
Wash Cells for guidance on choosing the appropriate wash option.

Labeling & Wash Buffer• Labeling or washing buffer for most sample types
is PBS + 1% BSA.
• For samples containing <70% viable cells, PBS +10% FBS can be used.
• BioLegend's Cell Staining Buffer can also be used
for labeling cells. However, this buffer may not be
optimal for all sample types. Cell Staining Buffer
should only be used for the labeling step. PBS + 1%
BSA should be used for the washing steps.

Sample Fixation• Consult Fixation of Cells & Nuclei for Chromium
Fixed RNA Profiling Demonstrated Protocol(CG000478) regarding fixing single cellsuspensions following cell surface protein labeling.

Sample Storage• Follow BioLegend's instructions if using
lyophilized antibody panel/cocktails regarding
reconstitution and labeling volumes.

• After labeling, samples may be fixed in smaller
batches and stored. If batching samples together,
it is recommended that samples with different
fixation times in one experiment are not mixed.

Centrifugation Conditions• Fixed samples can be stored either at -80°C for up
to 6 months or at 4°C for up to 7 days with appropriate storage reagents.

• Centrifugation speed and time depend upon thesample type.
• Larger or fragile cell types may require slower
centrifugation speeds.

Optimal Antibody Concentration• Consult Fixation of Cells & Nuclei for Chromium
Fixed RNA Profiling Demonstrated Protocol(CG000478) for details on storage conditions andreagents.

• The optimal concentration is 0.1 μg to 0.5 μg.
• Consult Quality Control of Cell Surface Protein
Labeling using Flow Cytometry Technical
Note (CG000231) for guidance on how to titrate
antibodies using flow cytometry for determining
optimal antibody concentrations.
• It is highly recommended to titrate antibodies
before use for optimal performance. Consult the
manufacturer's instructions for recommended
dilutions for each antibody. A dilution of 0.5 µg
per antibody for up to 1 x 106 cells is suggested as astarting point.

10xgenomics.com

7Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Cell Surface Protein
Labeling

Cell Surface Protein Labeling: Label Cells
Label Cells
Prepare Antibody Mix Supernatant and Flow Sorting Antibody Pool (if performing flow sorting) as described in Buffers – Preparation.

Add Antibody Mix
Supernatant*
Fc blocked Add PBS + 1% BSA
cells
Gently pipette mix

Labeledcells

Incubate for
30 min (4°C)

Low cellcounts or
viability?

No

Proceed to Wash
(1-Wash or 2-Wash)

Yes
Proceed to Sample Fixation,
Step c (CG000478)(No-Wash)*If performing cell sorting, add flow sorting Antibody Pool.

Follow manufacturer's instructions if using custom
conjugated antibodies. Consult Cell Surface Protein
Labeling for Single Cell RNA Sequencing Protocols
Demonstrated Protocol (CG000149) for details.
a. Add prepared Antibody Mix Supernatant to theFc blocked sample. If performing flow sorting,
add Flow Sorting Antibody Pool.
b. Add chilled PBS + 1% BSA to the cells to bring
the total volume to 100 μl. Gently pipettemix 10x (pipette set to 90 μl). For samples
containing <70% viable cells, chilled PBS + 10%
FBS can be used.

d. Proceed to appropriate Cell Surface Protein
Labeling: Wash Cells section for 1-Wash or
2-Wash Options
OR
Proceed directly to Sample Fixation, Step cof the Fixation of Cells & Nuclei for Chromium
Fixed RNA Profiling Demonstrated Protocol(CG000478) (No-Wash Option).

Sample fixation without washing is recommended
for samples with low inputs, low viability, or timesensitive samples where increased background isacceptable.

c. Incubate for 30 min at 4°C. If using fluorophoreconjugated antibodies, incubate without light
exposure.

10xgenomics.com

8Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Cell Surface Protein
Labeling

Cell Surface Protein Labeling:
Wash Cells
Using the appropriate wash option is critical for
obtaining high-quality data and minimizing
background signal. Choose a protocol based on thefollowing guidance. See the Appendix forsupplemental data on the different wash options.
No Wash Option

1-Wash Option• When a reduced number of wash steps is desired• Antibody background is expected to be moderate• Compatible with most antibodies that have aclear distinction between populations• Includes option for flow sorting enrichment• Samples with low viability or input counts ortime-sensitive samples

2-Wash Option (Recommended)• Antibody background is expected to be high• Recommended for most sample types• Compatible with antibodies that have clear
distinction between populations• Antibody background is expected to be low• Best quality Feature Barcode data

No Wash Option Protocol
No-Wash Option

Low cellcounts or
viability?

Yes

Proceed to Sample Fixation, Step c(CG000478)

Labeled Cells

a. Proceed immediately to:• Sample Fixation, Step c of the Fixationof Cells & Nuclei for Chromium Fixed RNA
Profiling Demonstrated Protocol (CG000478).

Step c: Add 1 ml Fixation Buffer to the labeled cells
and pipette mix 5x. Fixation Buffer preparation andfixation protocol are listed in Fixation of Cells & Nuclei
for Chromium Fixed RNA Profiling Demonstrated
Protocol (CG000478).
OR• Intracellular Protein Labeling10xgenomics.com

9Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Cell Surface Protein
Labeling

1-Wash Option Protocol
1-Wash Option

Low cellcounts or
viability?

Labeled Cells

No

No

Flow Sorting
Enrichment?

Add PBS + 1% BSA(1.4 ml) and count cells
Gently pipette mix
Proceed to Sample
Fixation, Step a(CG000478)

Yes
Resuspend in PBS + 2% FBS(include a dead cell marker)

Count
cells

Flow
Sortinga. OPTIONAL: For enrichment of labeled andviable cells by flow sorting:• Based on starting concentration andassuming ~50% cell loss, add an appropriate
volume chilled PBS + 2% FBS (including
a dead cell marker) to obtain a final cellconcentration of 5–10 x 106 cells/ml.
• Proceed to sorting (see Appendix for Flow
Sorting Guidance). After sorting, determine
cell concentration and viability using an
automated cell counter or a hemocytometer.

b. After resuspension, take an aliquot anddetermine cell concentration and viability
using an automated cell counter or ahemocytometer.
c. Proceed immediately to:• Sample Fixation, Step a of the Fixation ofCells & Nuclei for Chromium Fixed RNA Profiling Demonstrated Protocol (CG000478)
OR• Intracellular Protein LabelingIf not performing flow sorting enrichment:• Add 1.4 ml chilled PBS + 1% BSA to thelabeled cells. Gently pipette mix. For samples
containing <70% viable cells, chilled PBS +10% FBS can be used.

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10Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Cell Surface Protein
Labeling

2-Wash Option Protocol
2-Wash Option

Low cellcounts or
viability?

No
Add PBS + 1%
BSA (1.4 ml)
Gently pipette mix

Resuspend in PBS
400 rcf,
Remove+ 1% BSA (1.5 ml)
5 min, 4°C supernatantand count cells
Gently pipette mix

Proceed to Sample
Fixation, Step a(CG000478)

Labeled Cells

a. Add 1.4 ml chilled PBS + 1% BSA to thelabeled cells. Gently pipette mix. For samples
containing <70% viable cells, chilled PBS + 10%
FBS can be used.

d. Resuspend the cell pellet in 1.5 ml chilled
PBS + 1% BSA and place on ice. For samples
containing <70% viable cells, chilled PBS + 10%
FBS can be used.

b. Centrifuge cells at 400 rcf for 5 min (PBMCs)
at 4°C. Centrifugation speed and time depend
upon the sample type.

e. After resuspension, take an aliquot anddetermine cell concentration and viability
using an automated cell counter or ahemocytometer.

c. Remove the supernatant without touching thebottom of the tube to avoid dislodging the pellet.

Pellet(may notbe visible)

Leaving behind excess
supernatant (>30 μl) may
cause nonspecific binding,which may result inincreased backgroundreads during sequencing.

f.

Proceed immediately to:• Sample Fixation, Step a of the Fixationof Cells & Nuclei for Chromium Fixed RNA
Profiling Demonstrated Protocol (CG000478)
OR• Intracellular Protein Labeling10xgenomics.com

11Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Intracellular Protein Labeling
Intracellular Protein Labeling Protocol Overview

Label Cells

This protocol was optimized using human PBMCs
and the TotalSeqTM intracellular conjugates from
Biolegend and MultiPro Fixed Cell Immune
Profiling Antibody Cocktail, a pool of cell surface,intracellular, and isotype control antibodies, fromProteintech Genomics.
This protocol involves a short fixation before
staining and can be also be used with other cellsurface protein antibodies, provided they arecompatible with the brief fixation step. The labeledcells can be enriched by flow sorting (see Appendix
for Flow Sorting Guidance).

Use distinct and compatible antibody clones for
sorting. Optimize the working concentration of eachantibody used.

Intracellular Protein
Labeling

Fix Cells
Brief fixation

Fix Cells
CG000478

Buffers for Fixation - Prepare fresh
Quenching Buffer
Stock
Maintain at 4°C
Nuclease-free Water
Conc. Quench Buffer
8X(10x Genomics PN-2000516)
Thaw at room temperature.
Vortex and centrifuge briefly.
Total
-

Final
1X

Per Sample+ 10% (μl)
962.5
137.5

-

1,100.0

For Labeling Cells
Flow Sorting Antibody Pool Preparation• Add the appropriate/manufacturer's
recommended amount of fluorophore-conjugatedantibodies to a 1.5-ml microcentrifuge tube on
ice.
• Gently pipette mix and maintain at 4°C. Avoid
light exposure.

Buffers – Preparation
For Fixing Cells
Buffers for Fixation - Prepare fresh
Fixation Buffer
Stock
Maintain at room temperature
Nuclease-free Water
Conc. Fix & Perm Buffer(10x Genomics PN-2000517)
Thaw at room temperature.
Vortex, check for precipitation,and centrifuge briefly.
Formaldehyde*
Total

Final

Per Sample+ 10% (μl)

10X

1X

871.1
110.0

37%
-

4%
-

118.9
1,100.0*Formaldehyde should always be used with adequate
ventilation, preferably in a fume hood. Follow appropriate
regulations.
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12Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

Intracellular Antibody Mix Supernatant (Antibody+ Buffer) Preparation
Follow the manufacturer's recommendations forresuspension and aggregate removal. Generalguidance is provided below.

• Equilibrate the lyophilized panel vial(s) to room
temperature for 5 min.
• Place the lyophilized panel vial in an empty 2-ml
tube and centrifuge the vial at 10,000 rcf for 30
sec at room temperature.
a. Prepare Antibody Buffer in a Protein LoBind
tube.
Buffer for Labeling - Prepare fresh
Antibody Buffer
Stock
Final
For 1
Maintain at 4ºC
Sample +10% (μl)
RNase Inhibitor
1.1
40 U/μl
0.4 U/μl
Nuclease-free BSA

30%

7.55%

27.68

Tween 20

10%

0.2%

2.2

NP-40 or Nonidet
10%
0.5%
5.5
P40 SubstituteSalmon Sperm DNA 10 mg/ml 0.5 mg/ml 5.5
Dextran Sulfate
8 KDa
Monocyte Blocker

1%

0.025%

2.75

-

5 μl

5.5

FcX

-

5.5
43.27*

10X PBS

10X

5 μl
1X

Nuclease-free
Water*
If using lyophillized
antibody
Total

-

-

-

-

11.0

b. In case of lyophilized antibody panel:
Add 110 μl Antibody Buffer to rehydrate thelyophilized antibody panel. If using nonlyophilized antibody, directly proceed to step g.
c. Vortex the rehydrated antibody pool for 15 sec
and centrifuge at 10,000 rcf for 30 sec at room
temperature.
d. Incubate for 5 min at room temperature.
e. Vortex for 15 sec and centrifuge at 10,000 rcf for
30 sec at room temperature.
f.

Transfer the entire volume of the reconstituted
antibody pool to a new Protein LoBind tube andmaintain at 4°C.

g. Centrifuge at 14,000 rcf for 10 min at 4°C.
h. Transfer 100 μl supernatant (containing
antibody + buffer) to a new Protein LoBind tube
and maintain at 4°C.

For Washing Cells
Buffer for Washing - Prepare freshIntracellular Wash Stock Final
Buffer
Maintain at 4ºC
RNase Inhibitor
40 U/ 0.2 U/μl
μl
Tween 2010%0.1%
Nuclease-free BSA 30%
1%
10X PBS
10X
1X
Nuclease-free
Water
Total
-

For 1 Sample + 10%
(μl)
11.0
22.0
73.33
220.0
1873.67
2,200.0

110.0*Adjust water volume if:• adding reconstituted antibody pool derived from
non-lyophilized antibodies• adding fluorophore-conjugated antibodies for flow
sorting
Final buffer volume should not exceed 110 μl per
sample.

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13

Intracellular Protein
Labeling

If using a lyophilized antibody panel:

CG000529 | Rev C

Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Tips & Best Practices
Sample Storage• The intracellular labeling protocol includes abrief fixation (20 min) step.

• After labeling, samples may be fixed in smaller
batches and stored. If batching samples together,
it is recommended that samples with different
fixation times in one experiment are not mixed.

• Before starting the protocol, it is recommended
to test the antibodies by flow cytometry to ensurecompatibility with the fixation.

Optimal Antibody Concentration• It is highly recommended to titrate antibodies
before use for optimal performance. Consult the
manufacturer's instructions for recommended
dilutions for each antibody.

• Fixed samples can be stored either at -80°C for
up to 6 months or at 4°C for up to 7 days withappropriate storage reagents.
• Consult Fixation of Cells & Nuclei for Chromium
Fixed RNA Profiling Demonstrated Protocol(CG000478) for details on storage conditions andreagents.

• A dilution of 0.2 µg per antibody for up to 1 x 106
cells is suggested as a starting point.
• Consult Quality Control of Cell Surface Protein
Labeling using Flow Cytometry Technical
Note (CG000231) for guidance on how to titrate
antibodies using flow cytometry for determining
optimal antibody concentrations.

Antibody-Oligonucleotide Conjugation• Custom conjugated antibodies can also be usedwith this protocol. See Conjugation Guidance fordetails.
• Consult the Demonstrated Protocol Cell Surface
Protein Labeling for Single Cell RNA Sequencing
Protocols with Feature Barcode technology(CG000149) for guidance on antibodyoligonucleotide conjugation.

Antibody Quality• The specific antibody clones used needs to be ofhigh-quality and validated by flow cytometry.
• It may be helpful to use antibodies compatible
with flow cytometry.

Sample Fixation• Consult Fixation of Cells & Nuclei for Chromium
Fixed RNA Profiling Demonstrated Protocol(CG000478) regarding fixing single cellsuspensions following intracellular proteinlabeling.

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14

Intracellular Protein
Labeling

Brief Fixation & Antibody Compatibility

Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Intracellular Protein Labeling: Fix Samples
Fix Sample

Add Fixation
Buffer (1 ml)

Incubate at room
temperaturefor 20 min

RemovesupernatantProceed to Label Samples

Add Quenching
Buffer (1 ml)

a. Optional Step Cell Surface Protein Labeling
Skip this step and directly proceed to step b if
not utilizing the cell surface protein labelingsection in this document.

2000 rcf, 5 min,room temperature

Intracellular Protein
Labeling

Fc blocked orcell surface
protein labeled
cells

e. Remove the supernatant without disturbing thepellet.

• Perform cell surface protein labeling andwashing as described in the Cell Surface Protein Labeling section in this document.

Up to 30 µl of
supernatant canbe left behind• Centrifuge labeled & washed sample at 300400 rcf for 5 min (PBMCs/cell lines) at 4oC.
• Remove the supernatant without disturbing
the pellet.
• Proceed immediately to step b.
b. Add 1.0 ml room temperature Fixation Buffer
to the sample (Fc blocked or cell surface proteinlabeled) and pipette mix 5x.

f.

Add 1 ml chilled Quenching Buffer to thesample pellet and pipette mix 5x and keep on ice.

g. Proceed to the next step (Label Samples).

c. Incubate for 20 min at room temperature(20ºC).

DO NOT agitate or mix the sample during
incubation. To minimize variability for room
temperature fixations, incubation at controlled 20°C
temperature (e.g. with a thermomixer, heat block, orwater bath) is recommended.
d. Centrifuge at 2000 rcf for 5 min at room
temperature.
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Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Intracellular Protein Labeling: Label Samples
Label Samples
Permeabilization
Removesupernatant

Add Intracellular
Antibody Mix
Supernatant(antibody + buffer)
Incubate for
30 min (4°C)

Labeledcells

Add2000 rcf,
Intracellular 5 min, 4°C
Wash Buffer(1 ml)

Option 1

Removesupernatant

Option 2
if not sorting

Flow sorting*

Intracellular Protein
Labeling

Fixed cells

2000 rcf,
5 min, 4°C

Add
Intracellular
Wash Buffer(1 ml)

2000 rcf,
5 min, 4°C

Proceed to Sample
Fixation (CG000478)

Removesupernatant

Flow sorting**Flow sorting can be performed after the first wash OR after Sample Fixation (CG000478) depending on sample and antibody.

a. Centrifuge at 2000 rcf for 5 min at 4°C.
b. Remove the supernatant without disturbing thepellet.

OPTIONAL: For enrichment of cells by flow
sorting:• Proceed to flow sorting. See Post-Fixation
Flow Sorting for details. For collection ofsorted cells, use RNase-free reagents andsupplement all buffers with RNase inhibitors.

c. Add 100 μl Intracellular Antibody Mix
Supernatant (antibody + buffer) to the pellet
and gently pipette mix 10x.

• Flow sorting can also be performed after
step i (second fixation following theDemonstrated Protocol CG000478).
See Post-Fixation Flow Sorting for additional
information on collection.

d. Incubate for 30 min at 4°C. If using
fluorophore-conjugated antibodies, incubate
without light exposure.
e. Add 1.0 ml chilled Intracellular Wash Buffer tothe labeled cells. Gently pipette mix.
f.

Centrifuge cells at 2000 rcf for 5 min at 4°C.

g. Remove the supernatant without touching thebottom of the tube to avoid dislodging the pellet.

h. Repeat e-g one more time for a total of 2
washes.
i.

Proceed immediately to Sample Fixation,
Step c of the Fixation of Cells & Nuclei for
Chromium Fixed RNA Profiling Demonstrated
Protocol (CG000478).

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Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Intracellular Protein Labeling – Troubleshooting
Solution

Low cell viability when beginning the protocol

Perform dead cell removal (flow sorting or bead based)

High background

Remove as much residual buffer as possible during the centrifugation
steps, leaving no more than 30 μl buffer

Intracellular Protein
Labeling

Problem

Add single-stranded DNA binding protein (SSB) from bacteriophage
T4 (T4 Gene 32 Protein) to the antibody pool + antibody buffer if
the background signal is an issue. Use 8 μg SSB per μg of antibody
and add MgCl2 at 4 mM final concentration. It is recommended to use
flow cytometry to ensure that the SSB addition is not leading to a
reduction in antibody signal.
Poor intracellular protein labeling performance

Modify fixation time ranging from 15 to 30 min; 1 h fixation may becompatible as well, but will require testing with flow cytometry.
Lowering the fixation time may increase clumping while increasing
the time may reduce the antibody signal.
Modify NP-40 concentration ranging from 0.25% to 1%. Note that
≤0.25% NP-40 may not be enough for permeabilization, while >0.5%
may induce clumping.
Determine optimal antibody concentration using flow cytometry
Remove dextran sulfate in the blocking buffer, substitute with BSA
Increase antibody incubation time to >30 min

Poor Flex assay performance

Ensure that all buffers are made fresh and RNase-free
Ensure RNase inhibitors have been added to the Antibody Buffer andthe Intracellular Wash Buffer

Cell clumping

Perform dead cell removal (flow sorting or bead based)
Increase fixation time to up to 1 hr (testing antibody compatibility
with flow cytometry is recommended)
Reduce NP-40 concentration to <0.5%. Note that ≤0.25% NP-40 may
not be enough for permeabilization. Optimization and testing withflow cytometry is recommended.
Perform additional filtering steps, including before hybridization

Reads derived from antibodies targeting cell surface
antigens are a high percentage of the antibody library

Titrate surface antibodies to lower the amount of antibody added
Consult Quality Control of Cell Surface Protein Labeling using Flow
Cytometry Technical Note (CG000231) for guidance on how to titrate
antibodies using flow cytometry for determining optimal antibody
concentrations.

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17

Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Appendix

Post-Fixation Flow Sorting

Flow Sorting Guidance

Post fixation samples can be flow sorted foradvanced sample clean-up, as well as enrichment ofspecific populations. For samples undergoing
intracellular protein labeling, sorting can be
performed either after the first wash post labelingor after second fixation using the guidance
provided in Fixation of Cells & Nuclei for
Chromium Fixed RNA Profiling Demonstrated
Protocol (CG000478). Samples should be collected
in PBS + 1% nuclease-free BSA supplemented with
RNase inhibitor (Protector RNase inhibitor from
Sigma, PN-3335399001). A final concentration of
0.2 U/μl RNase inhibitor is recommended.

Enrich labeled cells using flow sorting prior to
library generation to enable identification of rare
subpopulations.
Pre-Fixation Flow Sorting
It is recommended to collect enriched cells in up to
20% FBS to maintain cell viability. Cells should becollected either in 20 �l volume in the collection
tube/plate (96-well plate) or in 300 �l volume in a
5-ml tube. Use an optimal buffer for fragile cells tomaintain a high cell viability.
Sort stream should be adjusted so that the celldroplet falls into the collection buffer. Sorted cells
should be counted and viability measured before
proceeding to the Fixation of Cells & Nuclei for
Chromium Fixed RNA Profiling Demonstrated
Protocol (CG000478). Cell loss during flow sorting
is common. Optimize the protocol steps
accordingly.

Consult 10x Genomics support website for more
information on postfixation cell sorting.

Once sorting is complete, proceed immediately
to Sample Fixation, Step a of the Fixation ofCells & Nuclei for Chromium Fixed RNA Profiling
Demonstrated Protocol (CG000478).

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18

Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

The Cell Surface Protein Labeling protocol provides three wash options after incubation with the antibody.
Using the appropriate wash option is critical for obtaining high-quality data and minimizing backgroundsignal. Supplemental data on the different wash options is provided in the following sections.

TotalSeqTM-C
50

anti-CD350
25

t-SNE_2

t-SNE_2

25

2-WashCD3E (FRP)

0
-25
-50
-50 -25

0

25

0
-25
-50
-50 -25

50

t-SNE_1

-25
-25

0

25

25

t-SNE_2

t-SNE_2

5
4
3
2
1
0

0

3
2

0

1

-25

0

-50
-50 -25

50

50

50

25

25

0
-25
-50
-50

-25

0

0

25

6
4
2
0

50

t-SNE_1

t-SNE_2

t-SNE_2

t-SNE_1

No Washanti-CD350

t-SNE_1

25

-50
-50

25

50

50

1-Wash0Expression Level

A.

25

50

No-Wash

1-Wash2-WashWash Steps

0
-25
-50
-50 -25

t-SNE_1

025

50

t-SNE_1

TotalSeqTM-B
anti-CD3

CD3E (FRP)30

t-SNE_2

2-Washt-SNE_230
0-30-50 -250

25t-SNE_1-50 -25

50

4
3
2
1
0

0
-25
-25

0

25

-25

0

0

25

50

t-SNE_1

64
2
0
No-Wash

1-Wash2-WashWash Steps

30

t-SNE_2

t-SNE_2

3
2
1

0
-50
-50 -25

50

30
0
-30
-50

anti-CD350

25

t-SNE_1

No Wash25

t-SNE_1

t-SNE_2

t-SNE_2

25

-50
-50

0

50

50

1-Wash0-30Expression Level

B.

-25

0

25

t-SNE_150

0-30-50 -250

2550

t-SNE_1

Figure 2. t-SNE plots of human PBMCs comparing staining for T cell marker CD3 across differing number of washes following antibody
incubation (left), showing equivalent data quality between TotalSeqTM-C (A) and TotalSeqTM-B (B) configuration of the same antibody
clone. Corresponding CD3E gene expression measured with Fixed RNA Profiling (FRP). Scale adjusted for better visualization with top 1%
of cells removed. Violin plot showing the expression of the anti-CD3 antibody Feature Barcode (right).
10xgenomics.com 19

Cell Surface Protein
Labeling

Supplemental Data on Different Wash Options – Cell Surface Protein LabelingDemonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Cell Surface Protein
Labeling

Supplemental Data on Different Wash Options – Cell Surface Protein Labeling contd.
TotalSeqTM-C

A.

50

25

25

t-SNE_2

0-25

TIGIT (FRP)

0-25-50 -250

25

50-50 -25

t-SNE_1

1.0

50

-25

0.5
0.0

25

t-SNE_2

t-SNE_2

1.5

0-50-50 -25

2.0
1.5
1.0
0.5
0.0

0
-25
-50

0

25

50

-50 -25

t-SNE_1
50

50

25

25

0
-25
-50
-50 -25

0

25

3
2
1
0

50

No-Washt-SNE_1

t-SNE_2

t-SNE_2

25

50

25

No Wash0

t-SNE_1

50

1-Washanti-TIGIT

-50

-50

Expression Level

2-Washt-SNE_2

anti-TIGIT
50

1-Wash2-WashWash Steps

0
-25
-50

0

25

50

-50 -25

t-SNE_1

025

50

t-SNE_1

TotalSeqTM-B

B.

anti-TIGIT

TIGIT (FRP)30

t-SNE_2

0-30-50 -25

0

25

50

0
-30
-50 -25

t-SNE_1

1.0

-25

0.5
0.0

0

25

25

t-SNE_2

t-SNE_2

1.5

0

-25
-50
-50 -25

50

25

50

3
2
1
0
No-Wash

1-Wash2-WashWash Steps

30

t-SNE_2

t-SNE_2

0

t-SNE_130
0-30-50 -25

2.0
1.5
1.0
0.5
0.0

0

t-SNE_1

No Washanti-TIGIT

50

t-SNE_1

25-50-50 -25

25

50

50

1-Wash0Expression Level

2-Washt-SNE_230

025

t-SNE_150

0-30-50 -250

2550

t-SNE_1

Figure 3. t-SNE plots of human PBMCs comparing staining for Natural Killer and T cell marker TIGIT across differing number of
washes following antibody incubation (left), showing equivalent data quality between TotalSeqTM-C (A) and TotalSeqTM-B (B)configuration of the same antibody clone. Corresponding TIGIT gene expression measured with Fixed RNA Profiling (FRP). Scale
adjusted for better visualization with top 1% of cells removed for each feature. Violin plot showing the expression of the anti-TIGIT
antibody Feature Barcode (right).
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Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Cell Surface Protein
Labeling

Supplemental Data on Different Wash Options – Cell Surface Protein Labeling contd.
TotalSeqTM-C

TotalSeqTM-B

Signal to Noise*(On-target cells / all other cells)

Signal to Noise*(On-target cells / all other cells)

Antibody

Comparison

No-Wash

1-Wash2-Wash

No-Wash

1-Wash2-Washanti-CD3
anti-CD4
anti-CD8
anti-TIGIT
anti-CD19
anti-CD15
anti-CD14
anti-CD11c

T cells / Other
T cells / Other
T cells / Other
T & NK cells / Other
B cells / Other
Monocytes / Other
Monocytes / Other
DCs & Monocytes / Other

12.44
9.92
3.2
1.14
27.11
2.9
6.62
13.5

15.72
10.71
5.81
1.4
39.67
4.51
7.4
12.89

16.99
9.36
6.32
1.54
45.61
4.61
8.26
13.07

13.09
7.61
3.11
0.85
16.94
1.94
6.45
14.07

14.89
7.17
4.94
0.89
23.26
2.24
7.53
12.1

17.58
8.05
5.93
1.1
35.48
2.88
9.77
14.81

*Ratio of mean expression level

Table 1. Signal to noise was generated by calculating the ratio of counts from a target group comprised of cell type(s) expected todisplay antibody signal (e.g. CD3 on T cells) to counts from a background group (e.g. CD3 on non-T cells).

Conclusion on Different Wash Options –Cell Surface Protein Labeling

Equivalent antibody Feature Barcode data quality
can be obtained regardless of format, either with
TotalSeqTM-C or TotalSeqTM-B antibody
oligonucleotide conjugates. However, only
TotalSeqTM-C enables sample multiplexing.
Choosing an appropriate wash protocol following
cell surface protein labeling is critical forexperimental success in the Chromium Fixed RNA
Profiling assay.

The data presented in this Demonstrated Protocol
show that a lower number of washes following celllabeling can be used for antibodies with distinct
positive and negative populations (Figures 2A and
2B, CD3 data). However, a lower number of washes
cause a reduction in separation between thepositive and negative populations (Figures 2A and2B, violin plot; Table 1, lower signal to noise forCD3, CD14, CD19, etc.). Feature Barcode data fromantibodies with poorer separation between thepositive and negative populations is adequate withthe 1-Wash protocol, but the No-Wash protocol is
not typically recommended due to poor signal tonoise (Figures 3A and 3B, TIGIT; Table 1, TIGIT).

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Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

References
1. Fixation of Cells & Nuclei for Chromium
Fixed RNA Profiling Demonstrated Protocol(CG000478)
2. Cell Surface Protein Labeling for Single Cell
RNA Sequencing Protocols with Feature
Barcode technology Demonstrated Protocol(CG000149)
3. Chromium Fixed RNA Profiling - Protocol
Planner (CG000528)
4. Chromium Fixed RNA Profiling Reagent Kits
for Singleplexed Samples with Feature Barcode
technology for Cell Surface Protein User Guide(CG000477)
5. Chromium Fixed RNA Profiling Reagent Kits
for Multiplexed Samples with Feature Barcode
technology for Protein using Barcode Oligo
Capture User Guide (CG000673)
6. Chromium Fixed RNA Profiling Reagent Kits
for Singleplexed Samples with Feature Barcode
technology for Protein using Barcode Oligo
Capture User Guide (CG000674)

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Demonstrated Protocol | Chromium Fixed RNA Profiling – Protein Labeling

CG000529 | Rev C

Document Revision Summary
Document Number

CG000529

Title

Cell Surface & Intracellular Protein Labeling for Chromium Fixed RNA
Profiling Demonstrated Protocol

Revision

Rev C

Revision Date

July 2024

Specific Changes

Added guidance on choosing the appropriate labeling protocol
Added Specific Reagents & Consumables list for Intracellular Protein
Labeling protocol
Added guidance on Intracellular Protein Labeling protocol

General Changes

Updated for general minor consistency of language and terms
throughout.

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