Jun 25, 2024
Protocol for harvesting and dissociating mouse brain neurons for single cell RNA Sequencing on the 10X Genomics platform
- Viktor Feketa1,
- Elena O. Gracheva1
- 1Departments of Cellular & Molecular Physiology and Neuroscience, Yale University School of Medicine, New Haven, CT, 06520, United States
Protocol Citation: Viktor Feketa, Elena O. Gracheva 2024. Protocol for harvesting and dissociating mouse brain neurons for single cell RNA Sequencing on the 10X Genomics platform. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74by3gwz/v1
Manuscript citation:
Isolation of neurons is based on the following published protocol with minor modifications: Vazirani, R. P., Fioramonti, X., Routh, V. H. Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing. J. Vis. Exp. (81), e50861, doi:10.3791/50861 (2013). https://www.jove.com/t/50861/membrane-potential-dye-imaging-ventromedial-hypothalamusneurons-from The protocol was further adapted for 10X Genomics platform according to the 10X Genomics "Cell Preparation Guide" (CG00053 Rev C): https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstratedprotocol-single-cell-protocols-cell-preparation-guide
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 05, 2022
Last Modified: June 25, 2024
Protocol Integer ID: 56579
Keywords: single cell RNA Sequencing, dissociating mouse brain neurons, 10X Genomics platform, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 020616
Abstract
Single-cell RNA sequencing has emerged as a powerful method to characterize gene expression on a single cell level. Producing useful data with this method critically relies on obtaining a suspension of dissociated cells with high concentration and viability from the tissue of interest. This protocol allows to isolate and dissociate mouse brain cellsinto a concentrated cell suspension that is compatible with the 10X Genomics library preparation and sequencing pipeline and enables capturing up to 10,000 single cells.
Attachments
338-741.pdf
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Guidelines
Isolation of neurons is based on the following published protocol with minor modifications:
Vazirani, R. P., Fioramonti, X., Routh, V. H. Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing. J. Vis. Exp. (81), e50861, doi:10.3791/50861 (2013).
The protocol was further adapted for 10X Genomics platform according to the 10X Genomics "Cell Preparation Guide" (CG00053 Rev C):
References:
1. Vazirani, R. P., Fioramonti, X., Routh, V. H. Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing. J. Vis. Exp. (81), e50861, doi:10.3791/50861 (2013).
Materials
Required custom equipment:
1. Oxygenation system, which consists of a gas tank with 95% O2/5% CO2 gas mixture and 3 tubing lines to provide continuous oxygenation and supply the gas to 1) a bottle with Brain Perfusion Solution, 2) reservoir (syringe) of the cardiac perfusion system, and 3) vibratome bath.
2. Cardiac perfusion system (gravity-driven), which consists of a 60-ml syringe secured ~65 cm above the working surface for mouse dissection with 1/4'' ID tubing (~1m long) ending with a Luer connection to a 20G needle. One of the oxygenation lines from the oxygenation system is inserted into the syringe reservoir of the perfusion system for oxygenation of the perfusion solution.
Materials:
Brain perfusion solution
Sucrose molecular biology gradeSigma AldrichCatalog #S0389
Sodium bicarbonateSigmaCatalog #S8875
GlucoseSigma AldrichCatalog #G5767-500G
Sodium L-ascorbateSigma AldrichCatalog #A4034
1 M Magnesium Chloride (MgCl2)Sigma AldrichCatalog #M8266
Sodium Pyruvate (100 mM)Thermo Fisher ScientificCatalog #11360070
Sodium chlorideSigmaCatalog #S5886
Media
L-( )-Lactic acidSigma AldrichCatalog #L1750
Glutamax (100x)Gibco - Thermo FischerCatalog #35050-061
B-27™ Supplement, minus insulinThermo FisherCatalog #A1895601
Penicillin/StreptomycinThermo Fisher ScientificCatalog #Invitrogen 15140-122
Bovine Serum AlbuminSigma AldrichCatalog #A9418
Labware
Equipment
15 mL Polystyrene Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile, 50/Rack, 500/Case
NAME
Polystyrene Centrifuge Tube
TYPE
Falcon®
BRAND
352099
SKU
LINK
Equipment
CELL CULTURE FLASK, 50 ML, 25 CM², PS
NAME
CELL CULTURE FLASK
TYPE
CELLSTAR® TC
BRAND
690160
SKU
LINK
10cc syringeBdCatalog #309604
Equipment
Syringe Filter Unit, 0.22 µm, mixed cellulose esters, 33 mm, ethylene oxide sterilized
NAME
sterile syringe filter
TYPE
Millex-GS
BRAND
SLGS033SS
SKU
LINK
Equipment
VWR® Disposable Pasteur Pipets, Glass
NAME
Disposable Pasteur Pipets
TYPE
VWR®
BRAND
14673-043
SKU
LINK
DNA LoBind Tube 2.0 mLEppendorfCatalog #022431048
Nalgene™ Rapid-Flow™ Sterile Disposable Filter Units with PES Membrane, 1000mL, Pore Size: 0.20µm, 90mm membraneThermo FisherCatalog #567-0020
Equipment
PYREX® 100x15 mm Petri Dish with Cover
NAME
Petri Dish with Cover
TYPE
PYREX®
BRAND
3160-101
SKU
LINK
MACS SmartStrainers 30umMiltenyi BiotecCatalog #130-098-458
Equipment
Equipment
Leica VT1200 - Semi-Automatic Vibrating Blade Microtome
NAME
Vibrating Blade Microtome
TYPE
Leica VT1200
BRAND
VT1200
SKU
LINK
Equipment
Centrifuge 5810 R
NAME
Plate Centrifure
TYPE
Eppendorf
BRAND
022625101
SKU
Equipment
Centrifuge
NAME
Bench centrifuge
TYPE
Eppendorf
BRAND
5424
SKU
Equipment
VWR® Shaking Water Baths
NAME
Shaking Water Baths
TYPE
VWR®
BRAND
10128-126
SKU
LINK
Equipment
OsmoTECH® Single-Sample Micro-Osmometer
NAME
Micro-Osmometer
TYPE
OsmoTECH®
BRAND
Osmotech
SKU
LINK
Equipment
Bright-Line™ Hemacytometer
NAME
Hemacytometer
TYPE
Bright-Line™
BRAND
Z359629
SKU
LINK
Surgical tools
Equipment
Surgical Scissors - Sharp-Blunt
NAME
Surgical Scissors
TYPE
Surgical Scissors
BRAND
14001-18
SKU
LINK
Equipment
Student Fine Scissors Straight 11.5cm
NAME
Fine Scissors
TYPE
Student Fine
BRAND
91460-11
SKU
LINK
Equipment
Graefe Forceps
NAME
Forceps
TYPE
Graefe
BRAND
11051-10
SKU
LINK
Equipment
Hippocampal Tool
NAME
Tool
TYPE
Hippocampal
BRAND
10099-15
SKU
LINK
Equipment
VWR® Flat/Spoon Spatulas, Stainless Steel
NAME
Spatulas
TYPE
VWR®
BRAND
82027-532
SKU
LINK
Other reagents/supplies
RNase AWAY™ Decontamination ReagentThermo FisherCatalog #10328011
Trypan Blue Solution, 0.4%Thermo FisherCatalog #15250061
1 ml tuberculin syringesBD BiosciencesCatalog #REF 309659
27G needlesCatalog #305109
Single-edge industrial razor bladesVwrCatalog #55411-050
| A | B | C | |
| Item | Supplier | Catalog No. | |
| Brain perfusion solution | |||
| Sucrose | Sigma | S0389 | |
| Potassium Chloride | JT Baker | 3040-01 | |
| Sodium Bicarbonate | Sigma | S8875 | |
| Sodium Phosphate, Monobasic, Monohydrate | JT Baker | 3818-01 | |
| Glucose | Sigma | G5767 | |
| (+)-Sodium L-ascorbate | Sigma | A4034 | |
| Calcium Chloride, Dihydrate | JT Baker | 1332-01 | |
| Magnesium Chloride Anhydrous | Sigma | M8266 | |
| Sodium Pyruvate solution (100 mM) | ThermoFisher | 11360-070 | |
| Sodium Chloride | Sigma | S5886 | |
| Media | |||
| Hibernate A (custom order with 2.5 mM Glucose; osmolarity 275 mOsm) | BrainBits | CUSTOM-HA | |
| Lactic acid | Sigma | L1750 | |
| GlutaMAX (100X) | ThermoFisher | 35050-061 | |
| B27 Supplement minus insulin 50x, 10 ml | ThermoFisher | A1895601 | |
| Penicillin-Streptomycin (10,000 U/mL) | ThermoFisher | 15140-122 | |
| Papain | Worthington | LS003124 | |
| DNAse | Worthington | LK003172 | |
| Bovine Serum Albumin | Sigma | A9418-5G | |
| Labware | |||
| Falcon 15 mL Polystyrene Centrifuge Tube | Corning | 352099 | |
| Cell culture flask, 50 ml | Greiner | 690160 | |
| 10 ml Syringes | BD | 309604 | |
| Millex-GS Syringe Filter Unit, 0.22 µm | EMD Millipore | SLGS033SS | |
| Glass Pasteur Pipets | VWR | 14673-043 | |
| DNA LoBind Tubes, 2.0 ml | Eppendorf | 22431048 | |
| Nalgene Rapid-Flow Sterile Single Use Vacuum Filter Units, 1000 ml, 0.2 µm pore size | ThermoFisher | 567-0020 | |
| PYREX 100x15 mm Petri Dish with Cover | Corning | 3160-101 | |
| MACS SmartStrainers, 30 µm | Miltenyi Biotec | 130-098-458 | |
| Equipment | |||
| Vibratome | Leica | VT1200 | |
| Centrifuge | Eppendorf | 5810 R | |
| Centrifuge | Eppendorf | 5424 | |
| Shaking Water Bath, 12L | VWR | 10128-126 | |
| OsmoTECH Single-Sample Micro-Osmometer | Advanced Instruments | Osmotech | |
| Bright-Line Hemocytometer | Sigma | Z359629 | |
| Surgical tools | |||
| Surgical Scissors, Sharp-Blunt, Straight | Fine Science Tools | 14001-18 | |
| Student Fine Scissors, Straight | Fine Science Tools | 91460 | |
| Graefe Forceps, Straight | Fine Science Tools | 11051-10 | |
| Hippocampal Tool (spatula) | Fine Science Tools | 10099-15 | |
| Flat/Spoon Spatula, Stainless Steel | VWR | 82027-532 | |
| Other reagents/supplies | |||
| RNase AWAY Decontamination Reagent | ThermoFisher | 10328011 | |
| Trypan Blue Stain (0.4%) | ThermoFisher | 15250-061 | |
| Krazy Glue All Purpose Precision Tip | Elmer’s Krazy Glue | ||
| 1 ml TB Syringe Slip Tip | BD | 309659 | |
| PrecisionGlide Needle 27G x ½ | BD | 305109 | |
| PrecisionGlide Needle 20G x 1 ½ | BD | 305179 | |
| Filter Papers Whatman 1 55mm | GE Healthcare | 1001-055 | |
| Double Edge Coated Blade Washed Version (for vibratome) | Electron Microscopy Sciences | 72000-WA | |
| Single Edge Industrial Razor Blade No.9 | VWR | 55411-050 | |
Safety warnings
Please follow the Safety Data Sheets (SDS) for all reagents for safe handling and safety hazards.
Ethics statement
Approval from an Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee should be obtained before performing these experiments. All animal procedures performed in relation to this protocol were performed in compliance with the Office of Animal Research Support of Yale University (protocol 2024-11497).
Part 1: Advance preparation of solutions
Part 1: Advance preparation of solutions
Prepare stock solutions of media supplements
Lactic acid: prepare 1 Molarity (M) (90 mg/mL ) solution of lactic acid in nuclease-free water and aliquot in50 µL aliquots. Store at -20 °C .
GlutaMAX: aliquot original GlutaMAX (200 millimolar (mM) ) solution into 100 µL aliquots. Store at-20 °C .
B27 (minus insulin) supplement: thaw original solution and aliquot into 700 µL aliquots. Store at -20 °C .
Prepare 1 L of Brain Perfusion Solution (composition: 2.5 millimolar (mM) KCl, 7 millimolar (mM) MgCl2, 1.25 millimolar (mM) NaH2PO4, 28 millimolar (mM) NaHCO3, 0.5 millimolar (mM) CaCl2, 7 millimolar (mM) glucose, 1 millimolar (mM) ascorbate, and 3 millimolar (mM) pyruvate in nuclease-free water). Adjust the osmolarity to ~300 mOsm using approximately 67 g/L sucrose. Oxygenate by bubbling with 95% O2 /5% CO2 for 00:15:00 and adjust the7.4 . Filter with a filter unit. Aliquot in 200 mL aliquots (each experiment will require approximately 200 mL of perfusion solution). Aliquots can be stored at-20 °C for up to 2 months. The day before experiment: thaw an aliquot of the Brain Perfusion Solution at4 °C Overnight .
We prepare the solution from the following specific components (for 1 L solution):
| A | B | C | D | E | |
| # | Component | Final Conc. (mM) | MW (g/mol) | Solid weight (mg/1000 mL) | |
| 1 | Sucrose | 96 | 342.3 | 67000 | |
| 2 | KCl | 2.5 | 74.55 | 186.4 | |
| 3 | NaHCO3 | 28 | 84.01 | 2352 | |
| 4 | NaH2PO4 * H20 | 1.25 | 137.99 | 172.5 | |
| 5 | Glucose | 7 | 180.16 | 1261 | |
| 6 | Sodium Ascorbate | 1 | 198.11 | 198.1 | |
| 7 | CaCl2 * 2H20 | 0.5 | 147.01 | 73.5 (or 0.5ml of 1 M stock) | |
| 8 | MgCl2 | 7 | 95.21 | 666.5 (or 7ml of 1 M stock) | |
| 9 | Sodium Pyruvate | 3 | 110.04 | 30 ml of 100 mM stock |
30m
The day before experiment, prepare a fresh 30 mL aliquot of Hibernate A media with 50 millimolar (mM) glucose, osmolarity of280 mOsm , and 100 U/ml penicillin-streptomycin.
Note
Note: target osmolarity of the working Hibernate A media is 280 mOsm . We order a custom formulation from BrainBits with 50 millimolar (mM) glucose and osmolarity nominally pre-adjusted to 275 mOsm . The actual measured osmolarity of the supplied media varies between ~270-280. Ordering it with the requested275 mOsm allows to adjust up to the target 280 mOsm . After receiving a new batch of media, we measure the original osmolarity, calculate approximate amount of NaCl to add to reach 280 mOsm , validate this amount empirically, and use the same amount for every experiment with a given batch/bottle of media to consistently obtain a working solution with the required target osmolarity of 280 mOsm .
In the sterile culture hood, transfer 30 mL of stock Hibernate A (pre-made by the supplier with 2.5 millimolar (mM) glucose and nominal osmolarity of275 mOsm ) to the 50 mL culture flask.
Add 300 µL of 100X Pen-strep stock solution to 30 mL Hibernate A aliquot.
Add NaCl (cell culture grade) to adjust osmolarity to 280 mOsm (~0 mg to 5 mg ).
Store media aliquot at 4 °C in dark until the day of experiment.
Fire-polish the tips of eight 9-inch glass Pasteur pipettes: 4 pipettes barely polished until the tips are no longer sharp (for cell transfers) and 4 pipettes for trituration with the following approximate tip opening diameters: 0.9 mm; 0.7 mm; 0.5 mm; 0.3 mm. The largest should be barely polished and the smallest should be about a third of that diameter.
Part 2. Experimental procedure on the day of experiment.
Part 2. Experimental procedure on the day of experiment.
Maintain RNAse-free conditions throughout the procedure. Wipe down all surfaces and tools with the "RNAse-away" reagent.
Thaw frozen aliquots of lactic acid, GlutaMax and B27 (minus insulin) supplements, vortex.
Prepare working solution of the Hibernate A media by adding the supplements for the following final working concentrations:
Lactic acid: 1 millimolar (mM) (add 30 µL of 1 Molarity (M) stock solution to the30 mL media aliquot).
GlutaMax: 0.5 millimolar (mM) (add 75 µL of 200 millimolar (mM) stck solution to the 30 mL media aliquot) .
B27: 2% (add 600 µL of the stock solution to the 30 mL media aliquot).
Mix media aliquot by inversion. Adjust pH to 7.4 using 1 Normality (N) NaOH.
Oxygenate the thawed 200 mL aliquot of the Brain Perfusion Solution with 95% O2/5% CO2 On ice for at least 00:15:00 .
15m
Prepare a 50 mL culture flask labeled "digestion" and 9 polystyrene 50 mL conical tubes labeled "harvest", "dissection", "papain", "DNAse", "BSA", "BSA filtered", "BSA centrifugation", "trituration", "cell suspension".
Distribute Hibernate-A media: transfer2 mL to the "harvest" tube,9 mL to "dissection" tube, 4 mL to "papain" tube, 5.5 mL to "DNAse" tube; 5 mL to "trituration" tube. Preserve the rest of Hibernate A media for next steps.
Place "harvest" and "dissection" tubes with media On ice near the animal dissection area.
Prepare RNA-se free area for animal dissection: wipe all surfaces and tools with "RNAse Away" reagent.
Prepare vibratome: get a new blade, wash with 70% ethanol followed by ultrapure water, load the blade in the vibratome blade holder.
Prepare area and tools for cardiac perfusion and animal dissection: bath for cardiac perfusion with absorbent pad, surgical tools, cardiac perfusion system with an oxygenation line inserted into perfusion reservoir.
Fill cardiac perfusion system with ultrapure water and let it run through to clean.
Add isoflurane to induction chamber for animal anesthesia.
Fill outer vibratome bath with ice and install on the vibratome.
Place 2 glass Petri dishes On ice .
Prepare oxygenation line (with 95% O2/5% CO2) to be later inserted into inner vibratome bath.
Place a mouse into induction chamber with isoflurane and wait until it stops breathing.
Take mouse out and verify depth of anesthesia by absence of response to toe pinch.
Pour ~30 mL of the ice-cold oxygenated Brain Perfusion Solution into reservoir of cardiac perfusion system just prior to dissection, fill tubing, and stop when about 20 mL of solution is left in the reservoir to be used for perfusion. Continue oxygenating the rest of the Brain Perfusion SolutionOn ice .
Perform mouse dissection.
Note
Note: the quicker cardiac perfusion is started, and then the brain extracted, the better for the survival of neurons.
Open abdominal cavity with scissors and peel back skin above ribcage, exposing diaphragm. Cut diaphragm and rib cage towards the forelimbs on both sides, then cut diaphragm along the edge of the rib cage to expose thoracic cavity. Be careful not to puncture the heart or big vessels. Peel back the ribcage towards the head, exposing the heart.
Insert the cardiac perfusion needle into left ventricle. Cut the right atrium with scissors.
Start the flow of perfusion system.
Perfuse about 15 mL of perfusion solution (~00:01:00 ) until the effluent is clear. Success of perfusion can be assessed by internal organs changing color to a lighter shade.
1m
Fill a petri dish On ice with ~ 15 mL of Brain Perfusion Solution.
Decapitate the mouse, Extract brain:
Cut skin above skull from caudal to rostral end and peel away to expose skull.
Make a midline cut with scissors in the skull towards the eye sockets. Use fine Graefe forceps to break and peel pieces of skull away from midline, exposing the brain.
Make a coronal cut with scissors between olfactory bulbs and the rest of the brain, and another cut between the brain and spinal cord to mobilize the brain.
Use a spatula to slightly lift the brain from the skull, use scissors to cut optical tracts, and finally extract the mobilized brain from the skull with a spatula (hippocampal tool) and push it into the Petri dish with the Brain Perfusion Solution.
Make a coronal cut between cerebellum and the rest of the brain, trying to make it as flat and perpendicular to the rostro-caudal axis of the brain as possible.
Note
This will make the brain sit flat on the vibratome stage and produce brain slices parallel to the coronal plane.
Lift the brain from the solution with a curved spatula and gently dab with filter paper to dry, especially the flat coronal aspect which will be glued down to the vibratome stage.
Put a little drop of super glue on the vibratome stage and spread it with a cotton tip to an area slightly larger than the brain.
Put the brain down on the stage area covered with glue, coronal aspect (now being the caudal end) down, rostral end up.
Mount the vibratome stage with the glued brain in the inner vibratome bath. About 00:00:10 after the brain attachment, fill the vibratome bath with the remaining oxygenated Brain Perfusion Solution to completely cover the brain in the bath.
10s
Rotate the stage of the vibratome with the brain to orient ventral (hypothalamic side) towards and dorsal cortex away from the blade.
Make sure the oxygenation line is inserted into vibratome bath and is turned on throughout the brain slicing.
Lower the vibratome blade into cutting position.
Using vibratome control pad, move the stage and blade as needed to perform the first cut close to the rostral end of the brain (facing up in the bath).
Start cutting 300 µm thick slices (vibratome settings: speed: 0.2 mm/sec, amplitude: 1 mm), observing the anatomical cues until the target area is reached. The level of bregma+1.0mm is reached approximately when left and right parts of corpus callosum meet in the middle.
After reaching the target area, cut two consecutive 300 µm thick slices to be collected.
Pour all 9 mL of Hibernate A media from the "dissection" tube into the second empty Petri dish On ice .
Transfer two target slices with a spatula from the vibratome bath into Petri dish with Hibernate A media.
Using 27G needles attached to 1 mL syringes used as cutting tools, dissect the target brain areas from slices. Further cut dissected pieces in two, to produce tissue pieces about 1x1 mm in size.
Collect tissue pieces with a glass pipette, transfer them to the "harvest" tube with Hibernate A media, and place the tube On ice .
Prepare digestion solution: add 80 U of stock papain suspension to the "papain" tube with 4 mL of media (for a final concentration of 20 U/ml ; calculate the volume of papain to get80 U beforehand based on the activity of specific batch, usually 65 µL to 85 µL ). Mix by inversion and place in 34 °C water bath. Check and mix by inversion every minute until the media is no longer cloudy (~00:04:00 ).
4m
During "papain" incubation, place the "harvest" tube with tissue pieces into 34 °C water bath.
Using 0.22-µm syringe filter, filter papain solution into a "digestion" culture flask.
Using glass pipette, transfer tissue pieces from "harvest" tube into "digestion" culture flask with papain. Shake "digestion" flask to make sure tissue pieces are not clumped together but are floating separately to ensure proper digestion.
Incubate "digestion" flask in a shaking water bath at 34 °C with shaking at 150 rpm, 00:30:00 .
Meanwhile, prepare media solutions for trituration:
Prepare DNAse solution: add 500 µL of Hibernate A media to a vial with DNAse solid, gently but thoroughly mix to dissolve completely (do not vortex, DNAse is sensitive to shear). Transfer 500 µL of dissolved DNAse to the "DNAse" tube with 5.5 mL of Hibernate A media (final volume 6 mL , final concentration of DNAse0.1 mg/mL ). Invert gently to mix (do not vortex).
Prepare BSA solution: weigh 160 mg of bovine serum albumin solid and add to the "bsa" tube with 2 ml of Hibernate A (final concentration: 8% BSA). Vortex for 30 sec. Using 0.22 µm syringe filter, filter BSA solution into another "bsa filtered" tube. Transfer 1 mL of filtered BSA to the "bsa centrifugation" tube.
Place 30 μm Miltenyi SmartStrainer onto "cell suspension" tube to collect and filter cell suspension.
When 00:30:00 digestion is done, transfer tissue pieces from "digestion" to the "trituration" tube using glass pipette. Invert once and let tissue pieces settle. Aspirate almost all media with glass pipette, leaving only tissue pieces.
30m
Add 3 mL of the trituration media ("DNAse" tube) to the "trituration" tube with tissue pieces. Triturate with the largest pipette (0.9 mm) 10 times (draw large volume to collect all tissue pieces with each trituration stroke) over approximately 00:00:30 . Wait 00:04:00 for tissue pieces to settle. Use the second-largest (0.7 mm tip opening) glass pipette to transfer 2 mL from the top of the cell suspension onto the cell strainer on top of "cell suspension" tube.
4m 30s
Add 2 mL of trituration media to the "trituration" tube. Triturate 10 times with the second-largest 0.7 mm pipette. Wait 00:03:00 for pieces to settle. Use the third (0.5mm) glass pipette to transfer the top 2 mL to the cell strainer/"cell suspension" tube.
3m
Add 1 mL of trituration media to the "trituration" tube. Triturate 5 times with the third 0.5 mm pipette. Wait 00:02:00 for tissue pieces to settle. Use the fourth 0.3 mm glass pipette to transfer all the remaining solution from the "trituration" to the cell strainer/"cell suspension" tube.
2m
Remove cell strainer from the "cell suspension" tube and layer all the solution from this tube onto the BSA layer in the "BSA centrifugation" tube using a glass pipette.
Note
Hold the target tube vertically, add drops gently and in a steady pace close to the solution surface to avoid disrupting the BSA layer and mixing solutions.
Centrifuge the "BSA centrifugation" tube1000 rpm, Room temperature, 00:05:00 . Using an Eppendorf 5810R swinging bucket centrifuge, this corresponds to 67 rcf. Set "break" setting to '0', i.e. no breaking.
5m
After centrifugation is done, carefully aspirate almost all media from the tube, leaving about 50 µL of solution above the cell pellet.
Add 950 µL of Hibernate A media to cell pellet. Resuspend cells with the glass pipette (gently pipette up and down 10 times).
Transfer all (~1 mL ) of the cell suspension from the "bsa centrifugation" tube to a new 2 mL Eppendorf LoBind tube.
Centrifuge at 300 rcf, 00:05:00 in a small tabletop centrifuge (Eppendorf 5424: 1787 rpm) at Room temperature .
5m
Carefully remove supernatant to leave ~50 µL of the solution, avoiding disrupting the cell pellet (it won't be visible) and creating bubbles.
Using glass pipette, carefully resuspend the cell pellet in the remaining ~50 µL of solution (pipette up and down about 10 times, avoid creating bubbles). This is the final cell suspension used for 10X library preparation. Place and store tube On ice until starting the 10X single cell protocol.
To determine the volume of solution to use for library preparation, determine cell concentration by counting cells using a hemocytometer. Transfer a 10 µL aliquot from the final cell suspension to a new2 mL LoBind tube. Add 10 µL of Trypan Blue stain. Mix gently with a pipette. Pipette 10 µL into a hemocytometer chamber. Count the number of live (transparent) and dead (blue) cells under the microscope. In case of harvesting primary and secondary motor areas from two 300 µm thick slices (4 tissue pieces ~1x2 mm in size), the expected cell concentration is ~1,000-2,000 cells/µl, expected viability is ~85%. To recover 10,000 cells in 10X protocol, the optimal concentration is 700-1,200 cells/µl. If obtained cell concentration is much higher than that, dilute cell suspension to the desired concentration by adding appropriate volume of Hibernate A media.
Proceed with the 10x Genomics Single Cell Protocol.
Note
In our case, the samples are delivered to the Keck Biotechnology Resource Laboratory/Yale Center for Genome Analysis at Yale University for further processing.
Expected results
Expected results
In case of harvesting primary and secondary motor cortex areas from two 300 µm thick slices (4 tissue pieces ~1x2 mm in size), the expected cell concentration is ~1,000-2,000 cells/µl, expected viability is ~85%.
Figure 1 shows a representative image of the cell suspension loaded in hemocytometer for counting.
Note
Please note that this image was captured for demonstration purposes ~00:30:00 after cell isolation, which resulted in a much higher number of dead cells (stained blue) than fresh preparation to be used in an actual experiment for downstream processing. Otherwise, the image gives a general idea of how the isolated cell suspension looks like during the hemocytometer counting step. This preparation had a concentration of ~1,600 cells/µl and viability of ~81% when assessed immediately after isolation.
Figure 1: Microscopic image of the cell suspension loaded in the hemocytometer during the cell counting step.
CITATION
Citations
Step 66
Vazirani, R. P., Fioramonti, X., Routh, V. H.. Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing
10.3791/50861 (2013).