CG000632 | RevB

DEMONSTRATED PROTOCOL

Isolation of Cells from FFPE Tissue Sections
for Chromium Fixed RNA Profiling
Introduction
This protocol outlines how to isolate cells fromformaldehyde fixed & paraffin embedded (FFPE)
Tissue Sections for use with Chromium Fixed RNA
Profiling workflow.
The protocol includes instructions for deparaffinizing and rehydrating the tissue sections, followed by
preparation and addition of a Dissociation Enzyme
Mix to the sections. The tissue sections can then be
dissociated using either a pellet pestle or a gentleMACS
Octo Dissociator. Once dissociated, cells are resuspended in the Tissue Resuspension Buffer or
Quenching Buffer prior to counting.
The minimum recommended cell inputs for
Chromium Fixed RNA Profiling hybridization
following this protocol are:• 400,000 cells for singleplex hybridization (Probe
Hybridization step 1.1d in User Guide CG000477*)

When following User Guide CG000477, for steps
2.1g-2.1j, perform one extra 0.5 ml wash for a totalof three 0.5 ml post-hybridization washes.
• 100,000 cells per Probe Barcode for multiplex
hybridization (Probe Hybridization step 1.1d inUser Guide CG000527)
Performing a pilot multiplex assay run (4 rxns with
4 Probe Barcodes) is recommended prior to committing to larger studies.
*Using cells derived from FFPE tissue sections with Feature Barcode
technology for cell surface protein detection is not supported.

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For optimal assay results, loading 10,000 cells per
Probe Barcode is recommended when performing
the singleplex assay or when multiplexing 4 samples.
For multiplexing 16 samples, loading 8,000 cells per
Probe Barcode is recommended (128,000 is the
maximum cells recovery per GEM reaction).
The protocol was demonstrated using FFPE tissue
blocks ranging 1-10 years in age. Each block may
yield different amounts of material and data
quality, depending on age, tissue type, pre-fixation
tissue quality, tissue density, size/area of tissue inthe scrolls, and other factors.

Additional GuidanceThis protocol was demonstrated using two or more
25 µm or 50 µm tissue sections from a diverse
spectrum of tissue types. For human tissue, it is recommended to use two or more 25 µm sections andfor mouse tissue, it is recommend to use two or more
50 µm sections. Some tissue types require morethan two sections to yield enough cells. FFPE blockproperties will also impact yields. Note that poor
quality blocks will likely yield data that cannot beinterpreted accurately or salvaged. See Appendix forcell yields derived from tissue sections of indicatedtissue types. In rare instances, a single 25 µm or
50 µm section may also yield adequate cells.
Tissue and cells may carry potentially hazardous
pathogens. Follow material supplier recommendations and local laboratory procedures andregulations for the safe handling, storage anddisposal of biological materials.

Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

Specific Reagents & Consumables
Vendor

Item

Part Number

For Tissue Section Transfer & Deparaffinization
Millipore
Sigma
VWR
Corning

ThermoFisherXylene, Reagent Grade214736Ethyl Alcohol, 200 Proof,
anhydrous
Ethanol absolute ≥99.5%,
TechniSolv, pure (for Europe)
Phosphate-Buffered Saline,
1X without Calcium andMagnesium
15 ml PP Centrifuge Tubes

E7023

Nuclease-free Water (notDEPC-Treated)

AM993783813.360DP
21-040-CV

gentleMACS C Tubes

Protector RNase Inhibitor

3335399001

Albumin, Bovine Serum*, 10%126615
Aqueous Solution, Nuclease-Free
UltraPure Bovine Serum
AM2616Albumin* (BSA, 50 mg/ml)

ThermoFisherScientific*Choose either Millipore or Thermo Fisher Scientific BSA.
VWR

Tris Buffer, 1M sterile sol., pH 8.0 E199-100ML

If using Quenching Buffer:430791

If using gentleMACS Octo Dissociator
Miltenyi
BiotecMillipore
Sigma

10x
Conc. Quench Buffer**
2000516
Genomics**Included in the 10x Genomics Chromium Next GEM Single
Cell Fixed RNA Sample Preparation Kit, 16 rxns (PN-1000414);
also includes Enhancer (PN-2000482) if storing fixed cells.

130-093-237
For Sample Filtration
SysmexAdditional Materials

Miltenyi
BiotecDisposable plastic Pasteur pipette
Nuclease-free Water
Water bath
Centrifuge

Sterile Single-Pack CellTrics 04-004-2326
Filters***(use 30 µm)
MACS SmartStrainers*** (30 µm) 130-098-458

OR
Pre-Separation Filters***(30 µm) 130-041-407***Choose either Sysmex or Miltenyi Biotec filter.
For Tissue Section Dissociation
Millipore
Liberase TH
Sigma
Corning
RPMI5401151001

For Cell Counting

10-040-CV

Nexcelom
Biosciences

If using pellet pestle:
FisherScientificFisherScientific

RNase-Free Disposable Pellet 12-141-364
Pestles
BD Luer-Lok PrecisionGlide
14-823-37
Disposable Syringes
with Detachable Needles
OPTIONAL

If using gentleMACS Octo Dissociator:
Miltenyi
BiotecgentleMACS Octo Dissociator 130-096-427
with Heaters

If using Tissue Resuspension Buffer:
Refer to SDS and follow local and institutional guidelines
for proper handling and disposal of all chemicals.

ThermoFisherScientificViaStain PI Staining Solution
ViaStain AOPI Staining
Solution
Alternative to PI Staining
Solution
Cellaca MX High-throughput†
Automated Cell Counter
Countess II FL Automated
Cell Counter†
Countess II FL Automated
Cell Counting Chamber Slides
Ethidium Homodimer-1

CS2-01095mL
CS2-01065mL

MX-112-0127
AMAQAF1000
C10228
E1169†Choose either Countess or Cellaca.

This list may not include some standard laboratory equipment.

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2

Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

Step Selection Overview

Preparation - Buffers
All buffers should be prepared fresh.
Prepare Dissociation Enzyme Mix, incubate at 37°C
for 10 min before proceeding with dissociation.
For pestle-based protocol
Dissociation
Enzyme Mix

Stock Final

1 rxn
(µl)

4 rxn + 10%(µl)

Liberase TH (mg/ml)

5

1

210

924

RPMI

-

-

840

3696

1050

4620

Total Volume (µl)

For gentleMACS Octo Dissociator protocol
Dissociation
Stock Final 1 rxn 4 rxn + 10%
Enzyme Mix
(µl)
(µl)
Liberase TH (mg/ml)

5

1

420

1848

RPMI

-

-

1680

7392

2100

9240

Total Volume (µl)

Tissue section
For pestle
dissociation
protocolOR

For Octo
Dissociator
protocolStep 1
Transfer sections to
1.5-ml tube;
deparaffinize

Step 1
Transfer sections togentleMACS C tube;
deparaffinize

Step 2
Pestle dissociation of
FFPE tissue

Step 3Octo Dissociator for
FFPE tissue

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

Prepare either Tissue Resuspension Buffer or
Quenching Buffer. They can be used interchangeably.
Tissue Resuspension
Stock Final
Buffer (Maintain at 4oC)

1 rxn 4 rxn + 10%
(µl)
(µl)

PBS

1X

0.496X 248

Tris buffer (pH 8.0; mM)

1000

50

25

110

BSA (RNase free)

10%

0.02%

1

4.4

RNase Inhibitor (U/µl)

40

0.24

3

13.2

Nuclease-free Water

-

-

223

981.2

500

2200

Total Volume (µl)
OR
Quenching Buffer(Maintain at 4oC)
Nuclease-free Water-

1 rxn 4 rxn + 10%
(µl)
(µl)
437.5
1925

1X

62.5

Stock Final-

8X
Conc. Quench Buffer*(10x Genomics PN 2000516)

1091.2

275

Total Volume (µl)
5002200*Included in the 10x Genomics Chromium Next GEM Single Cell
Fixed RNA Sample Preparation Kit, 16 rxns (PN-1000414).
Ethanol: Prepare fresh 70% and 50% Ethanol(1 ml each/sample).

Refer to SDS and follow local and institutional guidelines
for proper handling and disposal of all chemicals.
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3Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

Protocol Overview: Pestle for Isolation of Cells from FFPE Tissue Sections
1. Tissue Section Transfer & Deparaffinization
The scroll illustration is an abstract representation;
scrolls will be curled in the tube during execution

Add Xylene (1 ml);incubate for 10 min

Transfer to 1.5-ml
tube, keeping
the scrolls intact

TissueSections (x2)

Repeat 2x

Remove without
breaking thescrolls

1.5 mltube

When ready fordissociating,remove PBS

Add PBS (1 ml);
maintain on ice

Remove without
breaking thescrolls

Add water(1 ml);
incubatefor 30 sec

Add 100%
Ethanol (1 ml);incubate for
30 secRemove without
breaking
the scrolls

Repeat sequentially(30 sec each):
100 % Ethanol (1 ml)
70% Ethanol (1 ml)
50% Ethanol (1 ml)

Proceed to Pestle Dissociation of
FFPE Tissue

2. Pestle Dissociation of FFPE Tissue

Using a pellet pestle,
dissociate thescrolls to smaller
piecesAdd Dissociation
Enzyme Mix (100 µl)

Add Dissociation
Enzyme Mix(900 µl) while
rinsing the pestle tip

Add Tissue
Resuspension
Buffer (0.5 ml);resuspend pellet
Count cells

850 rcf
5 min
RemovesupernatantIncubate in athermomixer at 800 rpm(45 min at 37°C)
Can also use water bath

Wash filter
with additional
PBS (1 ml)

Triturate by pipette(1 ml) mixing (10-20x)

Pass througha 30 µm filter
Rinse the tube
with PBS (1 ml)& pass throughthe filter

Proceed to Chromium Fixed RNA
Profiling protocols (see References)

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4Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

Protocol Overview: gentleMACS Octo Dissociator for Isolation of Cells from FFPE
Tissue Sections
1. Tissue Section Transfer & Deparaffinization
The scroll illustration is an abstract representation;
scrolls will be curled in the tube during execution

Transfer togentleMACS C
tube keeping thescrolls intact
TissueSections (x2)gentleMACS

Repeat 2X

Add Xylene (3 ml);incubate for 10 min

Remove without
breaking thescrolls

Add 100%
Ethanol (3 ml);incubate for
30 secC tubeWhenready fordissociating,remove PBS

Add PBS (1 ml);
maintain on ice

Removewithout
breaking
the scrolls

Add water(1 ml);
incubatefor 30 sec

Removewithout
breaking
the scrolls
Repeat sequentially(30 sec each):
100 % Ethanol (1 ml)
70% Ethanol (1 ml)
50% Ethanol (1 ml)

Proceed to Octo Dissociator for
FFPE Tissue

3. gentleMACS Octo Dissociator for FFPE Tissue

Secure cap; attach tube
to gentleMACS Octo;
run appropriate
Add Dissociation
programEnzyme Mix (2 ml)

300 rcf
1 min

Resuspend pellet
in supernatant;filter through a
30 µm strainer

Wash strainer
with PBS (2 ml)

850 rcf
5 min

Proceed to Chromium Fixed
RNA Profiling protocols(see References)

Count cells

Add Tissue
Resuspension
Buffer (0.5 ml);resuspend pellet

Removesupernatant

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5Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

Isolating Cells from FFPE Tissue Sections
This protocol was demonstrated using 25 µm or 50 µm FFPE tissue sections of various tissue types (see Appendix
for details; maximum of six 50 µm sections were dissociated). The cells derived from the FFPE sections are compatible with the Chromium Fixed RNA Profiling assay. The recommended starting cell count for the Chromium
Fixed RNA Profiling for the singleplex protocol (CG000477) is 400,000 cells and for the multiplex protocol(CG000527) is 100,000 cells.

1. Tissue Section Transfer & Deparaffinization
Before starting, carefully inspect the tissue block to gauge
the extent of dehydration. Face the tissue block and place forsufficient time in an ice water bath (block facing the water
surface) to ensure proper rehydration of the tissue block.
See representative images of scrolls derived fromwell rehydrated and less rehydrated blocks in the
Troubleshooting section.
Prepare the first section from the rehydrated tissue blockby setting microtome to 5 µm. Discard the first section andset the microtome for 25 µm or 50 µm sections.
The volumes added in steps 1b, 1e, and 1g are
based on the type of tube being used for the
protocol.
a. After discarding the first section, prepare
up to two 25 or two 50 µm sections from therehydrated tissue block and transfer into a
1.5 ml tube (or to a gentleMACS C Tube) while
keeping the scrolls intact. The scrolls in the
tube can be stored at 4°C for up to one week.

Ensure that the scrolls stay intact during transfer
and through steps 1a-1m. Add reagents along theside of the tube and not directly onto the scrolls.
Intact sections enable easier liquid aspiration andminimize tissue loss. If scrolls break, see guidancein Troubleshooting section. Proceed with theprotocol and based on cell yields process more
sections if needed.
Always use fresh/freshly prepared reagents for the
deparaffinization steps.
Ensure the scrolls are fully submerged in regents inthe following steps.
b. Add 1 ml xylene to the 1.5-ml tube (3 ml to
gentleMACS C Tube) and incubate for 10 min atroom temperature.

c. Using a Pasteur pipette, remove the liquid from thetube without breaking the scrolls.
d. Repeat steps 1b-1c twice (for a total of three xylene
washes).
e. Add 1 ml 100% ethanol to the 1.5 ml tube (3 ml
to gentleMACS C Tube) and incubate for 30 sec atroom temperature.
f.

Using a Pasteur pipette remove the liquid from thetube without breaking the scrolls.

g. Add 1 ml 100% ethanol (to both tube types) andincubate for 30 sec at room temperature.
h. Using a Pasteur pipette remove the liquid from thetube without breaking the scrolls.
i.

Add 1 ml 70% ethanol (to both tube types) andincubate for 30 sec at room temperature.

j.

Using a Pasteur pipette remove the liquid from thetube without breaking the scrolls.

k. Add 1 ml 50% ethanol (to both tube types) andincubate for 30 sec at room temperature.
l.

Using a Pasteur pipette remove the liquid from thetube without breaking the scrolls.

m. Add 1 ml nuclease-free water to the tube andincubate for 30 sec at room temperature.
n. Using a Pasteur pipette remove the liquid from thetube without breaking the scrolls.
o. Add 1 ml PBS and maintain on ice.
p. Proceed to either to Step 2 Pestle Dissociationof FFPE Tissue (sections are in 1.5 ml tube)
or to Step 3 gentleMACS Octo Dissociator for
FFPE Tissue (sections are in gentleMACS C
Tube).
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6Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

2. Pestle Dissociation of FFPE Tissue
Refer to Preparation - Buffers section to prepare
Dissociation Enzyme Mix. Incubate at 37°C for
10 min before proceeding with dissociation.
a. Remove the PBS from the tube without
breaking the scrolls.

Ensure no tissue pieces are stuck to the tube cap.
Centrifuge at 300 rcf for ~20 sec to remove pieces
stuck on the cap.
f.

Using a 1,000 μl pipette, triturate the tissue
pieces in the tube by pipetting 10-20x.

b. Add 100 μl Dissociation Enzyme Mix to the tube.
Tissue Scrolls

The progress of the dissociation can be monitored
by taking a 10 μl aliquot and counting. If thecell concentration is lower than recommended
for hybridization, additional dissociation maybe
required; recommend performing step g.

c. Using a 1.5 ml pellet pestle, dissociate thetissue scrolls breaking them to smaller pieces.

g. OPTIONAL: Aspirate and push the tissue
pieces and solution through a 23G needle 5x to
improve cell recovery.

		 Grasp the pestle between the thumb and fingers androtate the pestle (clockwise and counterclockwise
10-20X) inside the tube and go up and down with thescrolls trapped between the wall of the tube and thepestle for dissociation or until the scroll is broken into
similarly sized smaller pieces.
The pellet pestle can also be attached to acompatible cordless motor and operated inside thetube until the scroll is broken into similarly sized
smaller pieces.
d. Add 900 μl Dissociation Enzyme Mix to thetube while rinsing the pestle tip into the tube tocollect any additional tissue pieces sticking to
the pestle. Pipette mix.

h. Pass the suspension through a Pre‑Separation
Filter (30 μm) placed on a 5- or 15-ml tube
placed on ice.
i.

Rinse the original tube (step 2f) with 1 mlchilled PBS and rinse the 30 µm filter withadditional 1 ml chilled PBS to minimize cellloss. Collect the filtrate in the same tube as
step 2h.

j.

Centrifuge the cell suspension at 850 rcf at
4°C for 5 min.

Use of swinging-bucket rotor is recommended forhigher cell recovery.
k. Remove the supernatant without disturbing thepellet.

e. Incubate for 45 min at 37°C in a thermomixer
at 800 rpm. Alternatively, incubate in a 37°C
water bath, mixing by inversion every 15 minduring the incubation.

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7Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profilingl.

CG000632 | Rev B

Resuspend the pellet in 0.5 ml chilled Tissue
Resuspension Buffer or Quenching Buffer,pipette mix 5x, and maintain on ice.

m. Determine cell concentration of the fixedsample using an Automated Cell Counter(Countess II/Cellaca MX) or hemocytometer.
See Appendix for Fixed Cell Counting.

For accurate cell counting, it is strongly
recommended that the cell suspension be
stained with a fluorescent nucleic acid dye suchas Ethidium Homodimer-1 or PI Staining Solution
and counted using an automated fluorescent cellcounter.
n. Proceed immediately to appropriate
Chromium Fixed RNA Profiling protocols –Probe Hybridization step 1.1d (see References)or store according to Fixed Sample Storage
Guidance provided in Appendix.

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8Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

3. gentleMACS Octo Dissociator for FFPE
TissueRefer to Preparation - Buffers section to prepare
Dissociation Enzyme Mix. Incubate at 37°C for 10
min before proceeding with dissociation.

f.

a. Remove the PBS from the gentleMACS C Tube
without breaking the scrolls.

g. Rinse the original gentleMACs tube (step 3d)with 2 ml chilled PBS and use that rinse for an
additional wash of the 30 µm filter to minimize
cell loss. Collect the filtrate in the same tube as
step 3f.

b. Add 2 ml Dissociation Enzyme Mix to thegentleMACS C Tube and close securely.

Ensure the scrolls are fully submerged in reagentsin the following steps.
c. Place the tube in on the gentleMACS Octo
Dissociator , apply Heating units and run the
gentleMACS Program 37C_FFPE_1. Run time
~48 min. Ensure that the Octo Dissociatorblades are moving before walking away.

gentleMACS Program 37C_FFPE_1
1
2

temp ON
spin -20 rpm, 5' 0" (counterclock)3

loop 3X

4spin 20 rpm, 14' 0"

5spin 1700 rpm, 7"

6
7

spin 1700 rpm, 1"
spin -1700 rpm, 2" (counterclock)8

spin 1700 rpm, 1"

9spin 1700 rpm, 4"

10

end loop

11

end

Use of swinging-bucket rotor is recommended forhigher cell recovery.
h. Centrifuge the cell suspension at 850 rcf at
4°C for 5 min.
i.

Remove the supernatant without disturbing thepellet.

j.

Resuspend the pellet in 0.5 ml chilled Tissue
Resuspension Buffer or Quenching Buffer,pipette mix 5x, and maintain on ice.

k. Determine cell concentration of the fixedsample using an Automated Cell Counter(Countess II/Cellaca MX) or hemocytometer.
See Appendix for Fixed Cell Counting.

For accurate cell counting, it is strongly
recommended that the cell suspension be
stained with a fluorescent nucleic acid dye suchas Ethidium Homodimer-1 or PI Staining Solution
and counted using an automated fluorescent cellcounter.
l.

d. At the end of the run, detach the tube fromthe gentleMACS Octo Dissociator and visuallyinspect to ensure the scrolls have been
dissociated. If scrolls are not dissociated, seeguidance in Troubleshooting section.

Pass the suspension through a Pre‑Separation
Filter (30 μm) placed on a 15-ml tube placed on
ice.

Proceed immediately to appropriate
Chromium Fixed RNA Profiling protocols –Probe Hybridization step 1.1d (see References)or store according to Fixed Sample Storage
Guidance provided in Appendix.

e. Centrifuge at ~300 rcf for 1 min and resuspend
the cell pellet in the supernatant.

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9Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

Appendix
Ethidium Homodimer-1 and PI stained cells can becounted using either Countess II FL Automated
Cell Counter or 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 evenin the presence of sub-cellular debris. The optimalcell concentration for the Countess is 1,000-4,000
cells/μl. Refer to manufacturer's instructions fordetails on operations.
• Vortex Ethidium Homodimer-1, centrifuge
briefly, and dilute the concentrated stock as per
manufacturer's instructions (~1:100 dilution).

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.

• Aliquot 10 μl diluted Ethidium Homodimer-1 ina tube.

• Transfer stained sample to Loading Row ofCellaca plate.

• Gently mix the sample. Immediately add 10 μl
sample to 10 μl diluted Ethidium Homodimer-1.
Gently pipette mix 10x.

• For counting fixed samples, only use the PI(Propidium Iodide) channel. Refer to manufacturer's instructions for details.

• 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.

Samples stained with PI staining solution can also
be counted using Countess II FL AutomatedCell
Counter. See manufacturer's instructions fordetails.
When counting cells after dissociation, the suspension may include some debris. However, during theChromium Fixed RNA Profiling workflow, the posthybridization wash step will reduce the debris.
Representative images after dissociation and after
post-hybridization wash (ready for loading on to thechip for GEM generation) are shown in Figure 1A
and 1B respectively.

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10Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

Figure 1A: Representative Cells after Dissociation

CG000632 | Rev B

Figure 1B: Representative Cells after Post-Hybridization Wash

Cellaca Counter (brightfield)

Cellaca Counter (brightfield)

Cellaca Counter (fluorescent)Cellaca Counter (fluorescent)10xgenomics.com

11Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

Fixed Sample Storage Guidance

Post-Storage Processing

Fixed samples (dissociated cells resuspended in
Quenching Buffer or in Tissue Resuspension Buffer)
can be stored for short or long-term as described
below.

Samples may undergo a color change duringstorage (e.g. black, light gray, or green), however
this will not impact assay performance.

Short-term Storage at 4 C
oa. 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
fixed sample in Quenching Buffer or in Tissue
Resuspension Buffer. For example, add 50 μl
Enhancer to 500 μl fixed sample in Quenching
Buffer or in Tissue Resuspension Buffer. Pipettemix.
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.

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 0.5 ml 0.5X PBS +0.02% BSA (optionally supplemented with 0.2
U/μl RNase Inhibitor) and keep on ice.

Use RNase-free BSA at this step. See Specific
Reagents & Consumables for details.
d. Determine cell concentration of the fixed sample
using an Automated Cell Counter (Countess II/
Cellaca MX) or hemocytometer. See Counting
guidelines.
e. Proceed immediately to appropriate
Chromium Fixed RNA Profiling protocols (seeReferences).

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
fixed sample in Quenching Buffer or in Tissue
Resuspension Buffer. For example, add 50 μl
Enhancer to 500 μl fixed sample in Quenching
Buffer or in Tissue Resuspension Buffer. Pipettemix.
c. Add 50% glycerol for a final concentration of
10%. For example: add 137.5 μl 50% glycerol
to 550 μl fixed sample in Quenching Buffer orin Tissue Resuspension Buffer with Enhancer.
Pipette mix.
d. Store at −80oC for up to 6 months.
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12Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

Cell Yields from FFPE Tissue Sections
Listed below are the cell yields from FFPE tissue sections processed as described in this protocol. Based on
this information, approximate cell yield from a specific tissue type/section may be estimated. Note that thetissue density in combination with tissue cross section area (tissue volume) will impact final cell yields.

HUMAN

MOUSE

Tissue Type

Tissue State(Healthy or Diseased)

TissueNo. of Sections x
Cross Section
Thickness (μm)(x*y mm)Cell Yields
gentleMACS
Manual Protocol Octo Dissociator

Brain (Cerebellum) Healthy

6 x 50 µm

2 x 2 mm

1.88 x 106

1.24 x 106

Brain (Forebrain)

Healthy

6 x 50 µm

4 x 5 mm0.545 x 106

0.2 x 106

KidneyHealthy3 x 50 µm

5 x 9 mm

4.73 x 106

3.89 x 106

Liver

Healthy3 x 50 µm

6 x 9 mm

1.7 x 106

1.6 x 106

SpleenHealthy3 x 50 µm

1 x 9 mm5.15 x 106

4.865 x 106

Brain

Healthy2 x 25 µm

5 x 10 mm

0.4 x 106

0.36 x 106

Brain

Glioblastoma

2 x 25 µm

10 x 13 mm

0.77 x 106

0.76 x 106

Breast

Healthy2 x 25 µm

10 x 6 mm

0.04 x 106

0.02 x 106

2 x 25 µm

13 x 7 mm

1.47 x 106

1.87 x 106

2 x 25 µm

10 x 7 mm

1.2 x 106

BreastCervixInvasive Ductal
Carcinoma
Endocervical
Adenocarcinoma

1.7 x 106

Colon

Cancer, Colorectal

2 x 25 µm

10 x 9 mm

0.6 x 106

0.76 x 106

KidneyHealthy2 x 25 µm

7 x 6 mm0.36 x 106

0.53 x 106

Liver

Healthy2 x 25 µm

5 x 6 mm

0.38 x 106

0.61 x 106

Liver

Hepatocellular
Carcinoma

2 x 25 µm

5 x 9 mm0.56 x 106

0.67 x 106

Lung

Healthy2 x 25 µm

9 x 7 mm0.44 x 106

0.79 x 106

Lung

Cancer2 x 25 µm

9 x 8 mm

0.92 x 106

0.76 x 106

Lymph Node

Healthy2 x 25 µm

7 x 5 mm

2.34 x 106

2.65 x 106

Lymph Node

Diseased, Reactive

2 x 25 µm

10 x 12 mm

6.75 x 106

7.45 x 106

Ovary

Cancer2 x 25 µm

10 x 18 mm

2.2 x 106

1.92 x 106

Pancreas

Healthy2 x 25 µm

11 x 9 mm

0.38 x 106

0.58 x 106

Prostate

Cancer2 x 25 µm

9 x 9 mm

0.8 x 106

1.6 x 106

Skin

Malignant Melanoma

2 x 25 µm

4 x 4 mm0.22 x 106

0.23 x 106

10xgenomics.com

13

Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

Troubleshooting
Problem

Solution

Tissue block
not adequately
rehydrated

Soak in water longer, until
rehydrated

Tissue section
curl breaks during
deparaffinization

Ensure tissue blocks is adequately
rehydrated(see adjacent representative tissue
section images from well rehydrated
and less rehydrated tissue blocks)

Tissue section
curl breaks during
rehydration
Lower than expected
cell yields

DO NOT pipette directly on top of
the curls; pipette against the tube
wall
Centrifuge at 850rcf for 1 min prior
to reagent exchange to pellet tissue
pieces at the bottom of the tubes
Refer to Cell Yields from FFPE
Tissue Sections table whenplanning the experiment
If possible, repeat sample
preparation protocol with more
sections to increase yield
Lower cell input recommendation
for multiplex hybridization. Consider
pooling cells derived from tissues ofsimilar complexity
If executing probe hybridization
step with lower cell input, ensurethat no cells are removed during
post-hybridization washes byleaving behind slightly more buffer
in the tube during the washes

Debris/large chunks
during cell counting

Representative Tissue Section Images
From well rehydrated blocks

From less rehydrated blocks

Minimal cracks in section

Cracks in section

Intact curls

Broken curls

References
1. Chromium Fixed RNA Profiling Reagent Kit
for Singleplexed Samples with Feature Barcode
technology for Cell Surface Protein User Guide*(CG000477)
2. Chromium Fixed RNA Profiling Reagent Kit forMultiplexed Samples User Guide (CG000527)*Using cells derived from FFPE tissue sections with Feature Barcode
technology for cell surface protein detection is not supported.

Pass sample through a 30 µm filter
followed by rinsing the filter with
PBS to minimize cell loss
Remaining debris (that passes
through the 30 µm filter) will befurther reduced during the posthybridization wash step in thedownstream workflow (see Figure 1B)

Using gentleMACS Octo Dissociator
Intact scroll at the
Run a "spin only" program on theend of run or run fails Octo Dissociator with steps 4-9
midway(from the protocol in step 3C)
Octo Dissociatorwithout heated lids

Use a water bath and every 14 minuse the "spin only" program on the
Octo Dissociator with steps 4-9(from the protocol in step 3C)
10xgenomics.com

14

Demonstrated Protocol | Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

CG000632 | Rev B

Document Revision Summary
Document Number

CG000632

Title

Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling

Revision

Rev A

Revision Date

December 2022

Specific Change

Updated long-term fixed sample storage recommendation at −80°C to 6 months(page 12)General Changes

Updated for general minor consistency of language and terms throughout© 2023 10x Genomics, Inc. (10x Genomics). All rights reserved. Duplication and/or reproduction of all or any portion of this document without the express written consent of 10x
Genomics, is strictly forbidden. Nothing contained herein shall constitute any warranty, express or implied, as to the performance of any products described herein. Any and all warranties applicable to any products are set forth in the applicable terms and conditions of sale accompanying the purchase of such product. 10x Genomics provides no warranty andhereby disclaims any and all warranties as to the use of any third-party products or protocols described herein. The use of products described herein is subject to certain restrictions
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