CG000478 | Rev C

DEMONSTRATED PROTOCOL

Fixation of Cells & Nuclei for Chromium Fixed RNA
Profiling
Introduction
Chromium Fixed RNA Profiling offers comprehensive scalable solutions to measure gene expression
in single cell and nuclei suspensions that are fixedwith formaldehyde. This protocol outlines how to
perform fixation on single cell and nuclei suspensions for use with Chromium Fixed RNA Profilingworkflow. This protocol also provides guidance onstorage of fixed cells and post-storage processing.
Prior to fixation, samples can also be labeled using
a specific protein binding molecule, such as an
antibody conjugated to a Feature Barcode oligonucleotide. Consult Demonstrated Protocol Cell
Surface Protein Labeling for Chromium Fixed RNA
Profiling with Feature Barcode technology(CG000529) for guidance.

Additional GuidanceThis protocol was demonstrated using primarycells (including peripheral blood mononuclear cells PBMCs), dissociated tumor cells (DTCs), cell lines(including Jurkat, K562, 293T), and nuclei suspensions. Optimize this protocol (e.g., centrifugation
conditions) based on sample type.
Cells carry potentially hazardous pathogens.
Follow material supplier recommendations andlocal laboratory procedures and regulations forthe safe handling, storage, and disposal of biological materials.

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Specific Reagents & Consumables
Vendor

Item

Part Number

For Cell Thaw & Sample Fixation
10x
Genomics

Chromium Next GEM Single
Cell Fixed RNA Sample
Preparation Kit
Millipore
Bovine Serum Albumin In
Sigma
DPBS (10%) Alternative to
Thermo Fisher product
ThermoUltraPure Bovine Serum
FisherAlbumin (BSA, 50 mg/ml)
Scientific
Alternative to Millipore Sigma
product
Formaldehyde (37% by
Weight/Molecular Biology),
Fisher BioReagents
Nuclease-free Water (notDEPC-Treated)
Miltenyi
MACS BSA Stock Solution
BiotecAlternative to Thermo Fisher
product
Corning
Phosphate-Buffered Saline,
1X without Calcium andMagnesium*Corning RPMI 1640 1X with
L-Glutamine
VWR*Seradigm Premium Grade
Fetal Bovine Serum (FBS)**Only needed for cell thawing.

1000414

A1595

AM2616

BP531-25

AM9937130-091-376

21-040-CV

10-040-CM
97068-085

For Sample Filtration
SysmexSterile Single-Pack CellTrics 04-004-2326
Filters
Miltenyi
Pre-Separation Filters
130-041-407
Biotec(30 µm)Choose either Sysmex or Miltenyi Biotec filter.

Demonstrated Protocol | Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling

CG000478 | Rev C

Preparation – Buffers
Buffers for Fixation - Prepare fresh

For Cell Counting
Nexcelom
ViaStain PI Staining Solution
Biosciences ViaStain AOPI Staining
Solution
Alternative to PI Staining
Solution.

ThermoFisherScientificCS1-0109-5mL
CS2-0106-5mL†Cellaca MX High-throughput MX-112-0127
Automated Cell Counter
AMAQAF1000†Countess II FL Automated
Cell Counter Discontinued
Countess II FL AutomatedC10228Cell Counting Chamber Slides
Trypan Blue Stain (0.4%)
T10282Ethidium Homodimer-1

E1169†Choose either Countess or Cellaca.

Millipore
Sigma

ThermoFisherScientific

Glycerol, 99.5%, for molecularbiology, DNAse, RNAse andProtease free
Alternative to Millipore Sigma
product
Glycerol for molecularbiology, ≥99.0%
Alternative to Acros Organics
product
Albumin, Bovine Serum, 10%
Aqueous Solution, NucleaseFree
Protector RNase Inhibitor

327255000

VWR

Per Sample(μl)

Nuclease-free Water

-

-

791.9

Conc. Fix & Perm Buffer*(10x Genomics PN-2000517)

10X

1X

100

Formaldehyde

37%

4%

108.1

Stock

Final

Per Sample(μl)

Nuclease-free Water

-

-

875.0

Conc. Quench Buffer*(10x Genomics PN-2000516)

8X

1X

125.0

Quenching Buffer
Maintain at 4oC

Additional Buffers

UltraPure BSA (50 mg/mL)

AM2616

Buffers for Storage of Fixed Samples - Prepare fresh
50% Glycerol Solution

For long-term storage of fixed samples

G5516-100ML• Mix an equal volume of nuclease-free water and 99% Glycerol, Molecular Biology Grade.
• Filter through a 0.2 μm filter.
• Store at room temperature in 2-ml LoBind tubes.

1266153335402001

Additional Buffers
0.5X PBS + 0.02% BSA (maintain at 4oC)
For post-storage processing; use RNase-free BSA
0.5X PBS + 0.02% BSA

DNA LoBind Tubes 2.0 ml

022431048

ThermoMixer C

5382000023

Vortex Mixer

10153-838

This list may not include some standard laboratory
equipment.

For 4
For 1
samplessample
+ 10%
(μl)
(μl)

Stock

Final

Nuclease-free Water

-

-

493.0

2169.2

1X PBS

1X

0.5X

500.0

2200.0

RNase-free BSA

10.0

0.02

2.0

8.8

RNase Inhibitor

40.0

0.2

5.0

22.0

Maintain at 4oC

Additional Materials
Eppendorf

Final

Maintain at room temperature

PBS + 0.04% BSA (maintain at 4oC)

For Storage & Post-Storage Processing
Acros
Organics

Stock

Fixation Buffer* Included in the 10x Genomics Chromium Next GEM
Single Cell Fixed RNA Sample Preparation Kit, 16 rxns
(PN-1000414).
Formaldehyde should always be used with adequate ventilation, preferably in a fume hood. Follow appropriate
regulations.
All buffer preparations should be fresh.
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2

Demonstrated Protocol | Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling

CG000478 | Rev C

Tips & Best Practices
The following recommendations are critical for
optimal performance of the Chromium Fixed RNA
Profiling assay.

• After each buffer addition step, gently mix cells/
nuclei 5x, or until the pellet is completely resuspended, without introducing bubbles.

Sample Quality

Fixation Conditions• Use high quality single cell or nuclei suspensions
that can withstand the fixation steps.
• Perform pilot experiments to determine if thesample type is suitable for the fixation.
• Highly viable single cell or nuclei suspensions(>80%) will have the greatest sensitivity and cellrecovery. However, the Chromium Fixed RNA
Profiling assay is robust to samples at much lower
viability, with successful results demonstrated
even with low viability samples (50% or lower).
Low viability samples may have more variable
cell calling and lower sensitivity.
• Samples should have minimal debris for best
results; debris can have associated RNA that cancontribute to non-cell background.

• Fixation temperature and time depends upon thesubsequent use of the fixed sample.
Fixed Sample Use

Fixation Time & Temperature

Fixed sample to beprocessed immediately
Fixed sample to be
stored subsequently

1 h at room temperature (20oC)
16-24 h at 4oC

DO NOT mix samples with different fixation times
in one experiment.
• If planning to store the fixed samples, it isstrongly recommended to perform a 16-24 hincubation at 4oC and store the fixed samples at-80oC for best results.

Fixed Cell Counting

Cell/Nuclei Number• This fixation protocol requires ≥300,000 cells or≥500,000 nuclei.

Centrifugation & Pellet Resuspension• Use a swinging-bucket rotor for higher cell/nuclei
recovery.
• Centrifugation speed and time may need optimization depending upon the sample type.
• When working with samples with low cellnumbers (i.e. <500,000 cells), complete removalof the supernatant is not required. Up to 30 µl
supernatant may be left behind to optimize cellrecovery without significantly impacting assay
performance.

• Accurate sample counting is critical for optimal
assay performance.
• It is strongly recommended that the sample be
stained with a fluorescent dye such as Ethidium
Homodimer-1 or PI Staining Solution andcounted using an automated fluorescent cellcounter (Countess II Automated Cell Counter or a
Cellaca counter).
• See Appendix for details on fixed cell counting.

Fixed Sample Storage• Fixed cell or nuclei suspensions can be stored at
4oC for up to 1 week or at -80oC for up to 6 months
after resuspending in appropriate reagents.
• Sample storage and post-storage guidelines areprovided in the Appendix.

Up to 30 µl
supernatant canbe left behind

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3Demonstrated Protocol | Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling

CG000478 | Rev C

Protocol Overview : Sample Fixation
Fix sample

Cells/nuclei
for fixing

300-400 rcf,
5 min*, 4oC

Removesupernatant

Add Fixation
Buffer (1 ml)

Incubate at room
temperature for 1 h
or at 4oC for 16-24 h

850 rcf, 5 min,room temperature

Removesupernatant

Add Quenching
Buffer (1 ml)

Proceed to Chromium Fixed
RNA Profiling protocols (seeReferences)

Count fixedcells*For dissociated tumor cells, centrifuge at 150 rcf for 10 min at 4°C.

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4Demonstrated Protocol | Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling

CG000478 | Rev C

Fixation Protocol
Chromium Next GEM Single Cell Fixed RNA Sample
Preparation Kit, 16 rxns (PN-1000414) was used forsample fixation. This protocol is compatible withboth cell and nuclei suspensions.
This protocol has been demonstrated using
300,000-10x106 cells or 500,000-10x106 nuclei. Ifcell/nuclei number exceeds this recommendation,
additional Fixation Buffer will be needed.

Optional
Label cells with TotalSeq-B antibody. Refer to
Demonstrated Protocol Cell Surface Protein
Labeling for Chromium Fixed RNA Profiling forSingleplexed Samples with Feature Barcode technology (CG000529) for details. Optional cell surface
protein labeling must be completed prior to sample
fixation.

Thaw Cells
If using fresh cells or nuclei suspensions, directly
proceed to Sample Fixation.

f.

Remove the supernatant without disturbing thepellet.

Transfer pipettes may be used to remove supernatant
after centrifugation to minimize disturbance to thepellet.
g. Resuspend the pellet in 1 ml chilled PBS +0.04% BSA. Gently pipette mix and transfer to a
1.5-ml microcentrifuge tube.
h. Determine and record cell concentration andviability of the sample using an Automated
Cell Counter (Countess II/Cellaca MX) or
hemocytometer. If cell debris and large clumps
are present, pass sample through a Cell Strainer.
i.

Proceed to Sample Fixation

Sample Fixationa. Centrifuge sample at 300-400 rcf for 5 min(PBMCs/cell lines) or 150 rcf for 10 min(dissociated tumor cells) at 4oC.
b. Remove the supernatant without disturbing thepellet.

a. Pre-warm 10 ml media (RPMI + 10% FBS) forcell thawing.

c. Add 1 ml Fixation Buffer to the sample pellet
and pipette mix 5x.

b. Remove cryovials containing cryopreserved
cells from storage, thaw in the water bath at
37°C for 1-2 min. Remove from the water bathwhen a tiny ice crystal remains.

d. Incubate for 1 h at room temperature (20oC)
or for 16-24 h at 4oC . If planning to store
fixed samples, a 16-24 h fixation at 4oC is
recommended.

c. Add 1 ml pre-warmed media (RPMI + 10% FBS)
to the thawed cell vial.

DO NOT agitate or mix the sample during
incubation.

d. Pipette mix the cells and transfer to a 15-ml
conical tube containing 9 ml pre-warmed
media (RPMI + 10% FBS).
e. Centrifuge at 300-400 rcf for 5 min (PBMCs/celllines) or 150 rcf for 10 min (dissociated tumor
cells) at 4oC.

Use of swinging-bucket rotor is recommended forhigher cell recovery. Centrifugation speed and time mayneed optimization depending upon the sample type.

To minimize variability for room temperature fixations,incubation at controlled 20oC temperature (e.g. witha thermomixer, heat block, or water bath) is recommended. Fixation time and temperature should beconsistent across all samples in an experiment.
e. Centrifuge at 850 rcf for 5 min at room
temperature.
f.

Remove the supernatant without disturbing thepellet.
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5Demonstrated Protocol | Fixation of Cells & Nuclei for Chromium Fixed RNA Profilingg. Add 1 ml chilled Quenching Buffer to thesample pellet and pipette mix 5x and keep on
ice.
h. Determine cell concentration of the fixedsample using an Automated Cell Counter(Countess II/Cellaca MX) or hemocytometer.
See Appendix for Fixed Cell/Nuclei Counting.

For accurate cell counting, it is strongly recommended
that the cell/nuclei suspension be stained with afluorescent dye such as Ethidium Homodimer-1 or
AO/PI Staining Solution and counted using an
automated fluorescent cell counter.
i.

Proceed immediately to appropriate
Chromium Fixed RNA Profiling protocols(see References) or store the sample after
resuspending in appropriate reagents.

Samples can be stored at 4 C for up to 1 weekor at -80oC for up to 6 months, depending upon
the reagents used for storage. See Appendix for
guidance on storage and post-storage processing.
oAppendix
Fixed Sample Storage Guidance
Fixed samples can be stored for short or long-term.

Short-term Storage at 4oC
a. Thaw Enhancer (10x Genomics PN-2000482)
for 10 min at 65°C. Vortex and centrifuge
briefly. Keep warm and verify no precipitate
before use.

DO NOT keep the thawed reagent on ice, or thesolution will precipitate. Once thawed, Enhancer
can be kept at 42°C for up to 10 min.
b. Add 0.1 volume pre-warmed Enhancer to fixedsample in Quenching Buffer. For example, add
100 μl Enhancer to 1,000 μl fixed sample in
Quenching Buffer. Pipette mix.

CG000478 | Rev C

c. Store sample at 4oC for up to 1 week.

Long-term Storage at −80oC
a. Thaw Enhancer (10x Genomics PN-2000482)
for 10 min at 65°C. Vortex and centrifuge
briefly. Keep warm and verify no precipitate
before use.

DO NOT keep the thawed reagent on ice, or thesolution will precipitate. Once thawed, Enhancer
can be kept at 42°C for up to 10 min.
b. Add 0.1 volume pre-warmed Enhancer to fixedsample in Quenching Buffer. For example, add
100 μl Enhancer to 1,000 μl fixed sample in
Quenching Buffer. Pipette mix.
c. Add 50% glycerol for a final concentration of
10%. For example: add 275 μl 50% glycerol to
1,100 μl fixed sample in Quenching Buffer andEnhancer. Pipette mix.
d. Store at −80oC for up to 6 months.

If planning to store the fixed samples, it is strongly
recommended to perform a 16-24 h incubation at 4oC
and store the fixed samples at −80oC for best results.

Post-Storage Processing
Samples may undergo a color change duringstorage (e.g. black, light gray, or green), however
this will not impact assay performance.
If samples were stored at −80oC, thaw at room temperature until no ice is present.
a. Centrifuge sample at 850 rcf for 5 min at room
temperature.
b. Remove the supernatant without disturbing thepellet.
c. Resuspend cell pellet in 1 ml 0.5X PBS + 0.02%
BSA* (optionally supplemented with 0.2 U/μl
RNase Inhibitor) or Quenching Buffer and keepon ice.

*Use RNase-free BSA at this step. See Specific
Reagents & Consumables for details.
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6Demonstrated Protocol | Fixation of Cells & Nuclei for Chromium Fixed RNA Profilingd. Determine cell concentration of the fixed sample
using an Automated Cell Counter (Countess II/
Cellaca MX) or hemocytometer. See Fixed Cell/
Nuclei Counting.

CG000478 | Rev C

Fixed PBMCs Stained with Ethidium Homodimer-1

e. Proceed immediately to appropriate Chromium
Fixed RNA Profiling protocols (see References).

Fixed Sample Shipping Guidancea. Fixed samples resuspended in Quenching Buffer
supplemented with Enhancer can be shippedwith a cold pack. See Short-term Storage fordetails.
b. Fixed samples resuspended in Quenching Buffer
supplemented with Enhancer and Glycerol canbe shipped on dry ice. See Long-term Storage fordetails.

Fixed Cell/Nuclei Counting• Accurate sample counting is critical for optimal
assay performance.
• It is strongly recommended that the fixedsample be stained with a fluorescent dye such as
Ethidium Homodimer-1 or PI staining solution
and counted using an automated fluorescent cellcounter (Countess II Automated Cell Counter or a
Cellaca counter).

Counting using Ethidium Homodimer-1
This protocol provides instructions for counting
samples using Ethidium Homodimer-1 and theCountess II FL Automated Cell Counter (with RFP
light cube) to enable accurate quantification even in
the presence of sub-cellular debris. The optimal cellconcentration for the Countess is 1,000-4,000 cells/μl.
Refer to manufacturer's instructions for details on
operations.
• Vortex Ethidium Homodimer-1, centrifuge briefly,
and dilute the concentrated stock as per manufacturer's instructions (~1:100 dilution).
• Aliquot 10 μl diluted Ethidium Homodimer-1 in a
tube.
• Gently mix the sample. Immediately add 10 μl
sample to 10 μl diluted Ethidium Homodimer-1.
Gently pipette mix 10x.

• Transfer 10 μl sample to a Countess II Cell
Counting Slide chamber.
• Insert the slide into the Countess II FL Cell
Counter. Image the sample using the RFP setting
for fluorescent illumination and filtering.
Optimize focus and exposure settings.
• Confirm the absence of large clumps using thebright-field mode. Make sure the cell counter iscircling RFP positive cells. Note the RFP-positive
concentration. Multiply by dilution factor 2 todetermine cell concentration.
Samples stained with Ethidium Homodimer-1 canalso be counted using Cellaca counter. Refer tomanufacturer's instructions for details.

Counting using PI Staining Solution
This protocol provides instructions for counting
sample using PI staining solution and the Cellaca
Counter to enable accurate quantification even in
the presence of sub-cellular debris. The optimalcell concentration for the Cellaca Counter is 10010,000 cells/μl. Refer to manufacturer's
instructions for details on operations.
• Add 25 μl PI Staining Solution into Mixing Row ofCellaca plate• Gently mix the sample. If the sample is tooconcentrated, a 1:1 dilution in PBS can also be
prepared. For example, add 15 μl fixed cell suspension to 15 μl PBS.
• Add 25 μl sample to Mixing Row of plate containing PI Staining Solution. Gently pipette mix 8x.
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7Demonstrated Protocol | Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling• Transfer stained sample to Loading Row ofCellaca plate.
• For counting fixed samples, only use the PI(Propidium Iodide) channel.
Refer to manufacturer's instructions for details.

CG000478 | Rev C

Brightfield image from Automated Cell Counter –Samples Stained with Trypan Blue
Fresh PBMCs

Samples stained with PI staining solution can also
be counted using Countess II FL Automated Cell
Counter. Refer to manufacturer's instructions fordetails.

Counting using Trypan Blue (Only for
Debris-free Samples)

Fixed PBMCs

Debris-free samples (cells or nuclei suspensions)can also be counted using trypan blue. This
protocol provides instructions for counting sample
using trypan blue and a hemocytometer orCountess II Automated Cell Counter.
• Mix 1 part 0.4% trypan blue and 1 part sample.
• Transfer 10 μl sample to a Countess II Cell
Counting Slide chamber or a hemocytometer.
• Insert the slide into the Countess II Cell Counterand determine the cell concentration. Or if using
hemocytometer, count fixed cells by placing
hemocytometer under the microscope.
• The majority of fixed cells or nuclei suspensions
will be stained with trypan blue stain and appear
non-viable.

References
1. Chromium Fixed RNA Profiling Reagent
Kits User Guide for Multiplexed Samples(CG000527)
2. Chromium Fixed RNA Profiling Reagent Kits
User Guide for Singleplexed Samples with
Feature Barcode technology for Cell Surface
Protein (CG000477)

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8Demonstrated Protocol | Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling

CG000478 | Rev C

Document Revision Summary
Document Number
Title
Revision
Revision Date
General Changes

CG000478
Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling
Rev C
February 2023

Specific Changes

Added Quenching Buffer for resuspension of samples during post-storage
processing.

Updated for general minor consistency of language and terms throughout

Removed −20oC from long-term storage.
Included table for the preparation of 0.5X PBS + 0.02% BSA.

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