Dec 19, 2019
MAINTENANCE CULTURE OF iPSCs (Basic Protocol 1)
- Michael S. Fernandopulle1,
- Ryan Prestil1,
- Christopher Grunseich1,
- Chao Wang2,
- Li Gan2,
- Michael E. Ward1
- 1National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland;
- 2Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
External link: https://doi.org/10.1002/cpcb.51
Protocol Citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. MAINTENANCE CULTURE OF iPSCs (Basic Protocol 1). protocols.io https://dx.doi.org/10.17504/protocols.io.48vgzw6
Manuscript citation:
Fernandopulle, M. S., Prestil, R., Grunseich, C., Wang, C., Gan, L., & Ward, M. E. (2018). Transcription-factor mediated differentiation of human iPSCs into neurons. Current Protocols in Cell Biology, e51. doi:https://doi.org/10.1002/cpcb.51
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: July 07, 2019
Last Modified: December 19, 2019
Protocol Integer ID: 25589
Keywords: i3LMN, i3Neurons, iPSC, iPSC-derived neurons, transcription factor-mediated differentiation
Abstract
Human iPSCs are an ideal system for studying human biology due to their rapid proliferation, genomic stability, and ability to differentiate into many somatic cell types. Historically, specialized culture practices and costly reagents have hindered widespread adoption of iPSCs by the cell biology community. In recent years, however, development of new culture techniques and improved media formulations have dramatically simplified iPSC culture and reduced costs.
The protocols described in this unit are adapted from a collection of publications that establish optimal practices for the maintenance of human iPSC cultures (Beers et al., 2012; Ludwig et al., 2006; Chen et al., 2011). While these publications provide useful guidelines for the stem cell novice, here we distill the fundamental procedures necessary for maintaining iPSCs in a pluripotent state and highlight critical steps that may need to be optimized for individual applications. In practice, iPSC lines of interest are usually maintained in an undifferentiated state in small cultures (1 to 3 wells of a 6-well plate) to reduce reagent use before being expanded as needed for experimentation.
Essential 8 (E8) is the simplest defined medium for hiPSC culture. E8 may be prepared from its components by the consumer (Table 1; Chen et al., 2011) or purchased as a preformulated kit. Other commercially available media may be substituted, such as mTeSR1 or StemFlex. A volume of 12 ml of medium should be added to each 10-cm tissue culture dish or distributed evenly across each standard multiwell plate (i.e., 2 ml/well for a 6-well plate). E8 should be aspirated and replaced with fresh medium daily, although a double volume may be added at low confluency to permit an extra day of culture without medium changes. StemFlex and E8 Flex contain components that stabilize the recombinant growth factors present in the medium, permitting medium exchange every other day as a general practice. Some iPSC lines (e.g., WTC11) tolerate every-other-day medium changes of standard E8 medium without loss of pluripotency or cell death, further reducing costs of medium and consumables. Finally, mTeSR1 may promote cell survival in stressful conditions better than E8, especially for finicky iPSC lines, although supplementation with a ROCK inhibitor (RI) is also recommended in such scenarios. Use of standard E8 will be assumed throughout this basic protocol.
Additionally, this protocol uses Matrigel-coated tissue culture plates. Matrigel works well for iPSC culture and has been widely adopted for research applications. However, since Matrigel is derived from murine sarcoma cells, it is not chemically defined and exhibits batch-to-batch variability. Alternative defined coatings include recombinant laminin or vitronectin, although these substrates are typically more costly. Notably, downstream neural differentiation described in these protocols occurs in fully defined conditions (see Basic Protocols 5 to 8), so the choice of iPSC substrate is of minimal scientific importance to all but clinical-grade applications.
Attachments
Materials
- Matrigel, hESC-qualified (Corning, cat. no. 354277)
Corning® Matrigel® hESC-Qualified MatrixCorningCatalog #354277
- DMEM/F12 medium (Gibco, cat. no. 11320033)
DMEM/F-12Gibco - Thermo Fisher ScientificCatalog #11320033
- Human induced pluripotent stem cells (hiPSCs; e.g., WTC11, Corriell Institute, cat. no. GM25256)
hiPSCsCoriell InstituteCatalog #GM25256
- E8 medium (may be user-formulated per Table 1 or purchased pre-formulated as Gibco, cat. no. A1517001; may also be substituted with E8 Flex, Gibco, cat. no. A2858501; StemFlex, Gibco, cat. no. A3349401; or mTeSR1, STEMCELL Technologies, cat. no. 85850; or similar)
DMEM/F-12, HEPESThermo Fisher ScientificCatalog #11330032
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A8960
Sodium seleniteMerck MilliporeSigma (Sigma-Aldrich)Catalog #214485
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S3817
Sodium chloride Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7653
Sodium hydroxide Merck MilliporeSigma (Sigma-Aldrich)Catalog #71463
Hydrochloric acid solution 1.0 NMerck MilliporeSigma (Sigma-Aldrich)Catalog #H9892
Insulin solution humanMerck MilliporeSigma (Sigma-Aldrich)Catalog #I9278
Recombinant Human TGF-β1 (HEK293 derived)peprotechCatalog #100-21
Recombinant Human FGF-basic (154 a.a.)peprotechCatalog #100-18B
holo-Transferrin humanMerck MilliporeSigma (Sigma-Aldrich)Catalog #T0665
Note
E8 may be made as described here and filter-sterilized before use (can be formulated in bulk and stored at -80°C) or may be purchased commercially.
| Component | Amount | |
| DMEM/F12 with HEPES | 500 ml | |
| L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate | 32 mg (per 500 ml) | |
| Sodium selenite (dissolve 0.1 mg/ml in PBS; handle in fume hood) | 7 μg (70 μl) (per 500 ml) | |
| Sodium bicarbonate | 271.5 mg (per 500 ml) | |
| Sodium chloride | As needed to adjust osmolarity to 340 mOsm | |
| Sodium hydroxide (1 M) | As needed to adjust pH to 7.4 | |
| Hydrochloric acid (1 M) | As needed to adjust pH to 7.4 | |
| Aliquot and add fresh to each bottle: | ||
| Insulin (supplied at 1000×, store at 4°C) | 500 μl (per 500 ml) | |
| TGF-β1 (2 μg/ml in PBS; 1000×, store at −80°C) | 500 μl (per 500 ml) | |
| FGF-basic (100 μg/ml in PBS; 1000×, store at −80°C) | 500 μl (per 500 ml) | |
| Holo-transferrin (10.7 mg/ml in PBS; 1000×, store at −80°C) | 500 μl (per 500 ml) |
Table 1: Essential 8 Medium
- 70% ethanol
- Rho-associated protein kinase (ROCK) inhibitor Y-27632 (e.g., Tocris Bioscience, cat. no. 1254 or Selleck Chemicals, cat. no. S1049), reconstituted to 10 mM in PBS
Y-27632 dihydrochlorideTocrisCatalog #1254
- Phosphate-buffered saline (PBS) without calcium or magnesium (e.g., Gibco, cat. no. 10010049)
Phosphate-buffered saline (PBS) without calcium or magnesium Gibco - Thermo Fisher ScientificCatalog #10010049
- 0.5 mM EDTA in PBS (diluted from Gibco, cat. no. AM9260G or Sigma, cat. no. E6758; may also be purchased as Versene, Gibco, cat. no. 15040066)
EDTA (0.5 M, pH 8.0, nuclease-free)Thermo Fisher ScientificCatalog #AM9260G
- Accutase (Gibco, cat. no. A1110501 or StemCell Technologies, cat. no. 07920)
StemPro™ Accutase™ Cell Dissociation ReagentThermo Fisher ScientificCatalog #A1110501
ACCUTASE™STEMCELL Technologies Inc.Catalog #07922
- Dimethylsulfoxide (DMSO; Sigma, cat. no. 472301)
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
- Fetal bovine serum (FBS), qualified, heat inactivated (Gibco, cat. no. 16140071)
Fetal Bovine Serum, qualified, heat inactivated, United StatesThermo Fisher ScientificCatalog #16140071
- Liquid nitrogen
- CoolRack M30 (BioCision BCS-108)
Equipment
CoolRack® M30
NAME
Sample Cooling Rack
TYPE
biocision
BRAND
BCS-108
SKU
LINK
- P2, P20, P200, and P1000 micropipettors and tips (e.g., Gilson)
- Sterile 5, 10, and 25-ml serological pipets (e.g., Corning, cat. no. 356543, 356551, and 357535, respectively)
Equipment
Falcon® 10 mL Serological Pipet
NAME
Serological Pipet
TYPE
Falcon
BRAND
356551
SKU
LINK
Equipment
Falcon® 25 mL Serological Pipet
NAME
Serological Pipet
TYPE
Falcon
BRAND
357535
SKU
LINK
- Sterile 15- and 50-ml polypropylene conical tubes (e.g., Corning, cat. nos. 352096 and 352070, respectively)
Equipment
Falcon® 15 mL High Clarity PP Centrifuge Tube
NAME
Centrifuge Tube
TYPE
Falcon
BRAND
352096
SKU
LINK
Equipment
Falcon® 50 mL High Clarity PP Centrifuge Tube
NAME
Centrifuge Tube
TYPE
Falcon
BRAND
352070
SKU
LINK
- Sterile polystyrene 10-cm tissue-culture dishes and 6-well, 12-well, and 24-well plates (e.g., Corning, cat. no. 353003, 353046, 353043, and 353047, respectively)
Equipment
Falcon® 100 mm TC-treated Cell Culture Dish
NAME
Cell Culture Dish
TYPE
Falcon
BRAND
353003
SKU
LINK
Equipment
Falcon® 6-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate
NAME
Cell Culture Plate
TYPE
Falcon
BRAND
353046
SKU
LINK
Equipment
Falcon® 12-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate
NAME
Cell Culture Plate
TYPE
Falcon
BRAND
353043
SKU
LINK
Equipment
Falcon® 24-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate
NAME
Cell Culture Plate
TYPE
Falcon
BRAND
353047
SKU
LINK
- Laminar flow biological safety cabinet (BSC)
- Vacuum aspirator and aspirating pipets (Fisher, cat. no. 1367820) or reusable Corning vacuum aspirator (cat. no. 4930) with disposable tips (e.g., Pure XLG pipet tips, Andwin Scientific, cat. no. 46600-020)
Equipment
Fisherbrand™ Disposable Borosilicate Glass Pasteur Pipets
NAME
Pasteur Pipet
TYPE
Fisherbrand
BRAND
1367820
SKU
LINK
Equipment
Corning® Vacuum Aspirator
NAME
Vacuum Aspirator
TYPE
Corning
BRAND
4930
SKU
LINK
Equipment
MBP PURE XLG 200 UL PIPET TIP
NAME
Pipet Tip
TYPE
Andwin Scientific
BRAND
46600-020
SKU
LINK
- Phase-contrast and fluorescent microscope with 4×, 10×, 20×, and Object Marker objectives (e.g., Nikon Eclipse Ti)
- Cell counting apparatus [hemacytometer or automated cell counter; also see Phelan & May (2015)]
- 50-ml, 250-ml, and 500-ml sterile filters, 0.2-μm pore (Millipore, cat. no. SCGP00525; Thermo, cat. no. 568-0020 and cat. no. 566-0020, respectively)
Equipment
Steriflip-GP
NAME
Sterile Centrifuge Tube Top Filter Unit
TYPE
Millipore
BRAND
SCGP00525
SKU
LINK
Equipment
Nalgene™ Rapid-Flow™ Sterile Disposable Filter Units with PES Membrane
NAME
Filter
TYPE
Thermo Scientific™
BRAND
568-0020
SKU
LINK
Equipment
Nalgene™ Rapid-Flow™ Sterile Disposable Filter Units with PES Membrane
NAME
Filter
TYPE
Thermo Scientific™
BRAND
566-0020
SKU
LINK
- 1.5-ml cryogenic tubes (Thermo, cat. no. 5000-1020)
Equipment
Nalgene™ General Long-Term Storage Cryogenic Tubes
NAME
Storage Tubes
TYPE
Thermo Scientific™
BRAND
5000-1020
SKU
LINK
- Microcentrifuge for 1.5-ml tubes
- Cryovial freezing container (e.g., CoolCell LX, BioCision, cat. no. BCS-405; or Mr. Frosty, Thermo, cat. no. 5100-0001)
Equipment
Cell freezing container
NAME
Freezing Container
TYPE
CoolCell LX
BRAND
BCS-405
SKU
LINK
Equipment
Mr. Frosty Freezing Container
NAME
freezing container
TYPE
ThermoFisher Scientific
BRAND
5100-0001
SKU
LINK
- Picking microscope inside sterile laminar flow enclosure (e.g., Etaluma LS620)
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Matrigel Coating
Matrigel Coating
Aliquotting concentrated Matrigel:
Note
Matrigel polymerizes rapidly at room temperature when concentrated, so it is imperative to aliquot stocks with pre-chilled tips and tubes and to thaw the concentrated stock solution on ice.
Gradually thaw a 5 mL bottle of Matrigel stock solution overnight at 0 °C by burying in ice in a Styrofoam container placed within a refrigerator. Additionally, pre-chill microcentrifuge tubes by placing in an aluminum cool rack on ice before use.
Before pipetting concentrated Matrigel into pre-chilled microcentrifuge tubes, chill a 1-ml pipet tip by pipetting ice-cold DMEM/F12 up and down several times, then immediately use this chilled tip to aliquot the Matrigel stock.
Aliquot 500 µL concentrated Matrigel into each microcentrifuge tube, and refreeze aliquots at -80 °C .
Making Matrigel coating solution:
Aliquot 50 mL of cold DMEM/F12 into a conical tube.
Using a P1000, pipet 1 mL of cold DMEM/F12 from the conical tube into the microcentrifuge tube containing 500 µL concentrated Matrigel stock. Pipet up and down several times, and then transfer what has thawed to the conical tube containing cold DMEM/F12.
Repeat until the frozen concentrated Matrigel has been completely transferred to the 50-ml conical tube containing DMEM/F12. Invert several times to mix.
Add one half of the normal culture volume of the Matrigel coating solution to the tissue culture surface.
Gently agitate plates to ensure full coverage.
Note
For example, add 1 mL per well of a 6-well plate.
Transfer plates to a 37 °C incubator.
Note
Plates may be used after 1 hr, but better long-term morphology typically results from overnight coating. It is also possible to prepare plates in bulk by adding additional DMEM/F12 to a full culture volume in order to prevent wells from drying. These plates may be stored in a 37 °C incubator or wrapped in Saran wrap or Parafilm and stored at 4 °C . Plates should be used within 2 weeks of preparation.
Aspirate Matrigel solution immediately before use and replace with culture medium and cells.
Thawing iPSCs
Thawing iPSCs
Note
Often, iPSCs are stored and distributed as frozen stocks, so thawing is the first procedure performed. Routine use of antibiotics in stem cell culture medium is strongly discouraged, since these compounds can interfere with cell biochemistry and differentiation potential.
Consequently, proper sterile technique is critical to prevent contamination, and cells received from other environments should be quarantined for at least two passages and tested for mycoplasma.
Prepare biological safety cabinet (BSC) with tube racks, DMEM/F12, P1000 tips, conical tubes, and culture medium.
Note
DMSO is toxic to cells at room temperature, so steps 2 to 5 should be completed as quickly as possible.
Transfer cryovial of hiPSCs from liquid nitrogen or dry ice and thaw in 37 °C water bath or bead bath.
Note
Thaw should be completed rapidly to limit exposure to DMSO. A small frozen core may remain, as pipetting and rinsing will complete thaw.
Sterilize cryovial by spraying with or dipping into 70 % ethanol and transfer into the BSC.
Pipet cell solution to new 15-ml conical tube, rinse cryovial twice with 1 mL DMEM/F12, and add each rinse to the tube.
Centrifuge tube 00:05:00 at 300 x g , Room temperature .
Note
Speeds of 200 to 300 × g are well tolerated by iPSCs. For the purposes of this protocol, 300 × g is recommended to maximize capture of small cell numbers. For standard procedures, 200 × g is recommended.
Aspirate supernatant, resuspend in culture medium supplemented with 10 micromolar (µM) Y-27632 ROCK inhibitor, and transfer to Matrigel-coated plate.
Note
Maintaining high cell density maximizes survival, so it is recommended to plate each vial (typically 1 × 106 cells) in one well of a 6-well plate. This may be modified depending on specific cell number or viability.
Return plate to 37 °C incubator and evenly distribute cells by gently shaking platefront-to-back and side-to-side.
Note
This procedure is critical any time cells are replated, and should be performed as soon as cells are transferred. Swirling or otherwise agitating culture medium before cells attach can cause higher cell densities in the middle of the well.
The next day, aspirate the medium and replace with fresh E8 culture medium (2 ml/well for 6-well dish). If colonies are small and/or if cell death is noted after the medium change, use of E8 with Y-27632 ROCK inhibitor may be necessary until colonies have expanded, after which inclusion of the ROCK inhibitor is not required.
Note
An advantage of using iPSCs as a model system is their rapid rate of proliferation; however, culture health is superior if cultures are only split to modestly low densities. Furthermore, cells will rapidly differentiate and die if allowed to grow into a monolayer. When colonies grow too large and/or begin to converge (approximately 80% confluency), they must be dissociated in order to maintain proper growth and pluripotency. Dissociating cells with EDTA is gentler and faster, and typically results in improved survival over enzymatic dissociation methods, making it ideal for general culture maintenance. EDTA acts by chelating the calcium necessary for cell attachment and transfers cells as small clumps, which promotes colony formation and growth. Alternatively, Accutase provides gentle enzymatic dissociation for iPSCs and should be used for any applications that require single-cell suspensions, such as for clonal derivation, cell counting, or flow sorting. Supplementation of culture medium with ROCK inhibitor is optional following EDTA treatment, but is required after Accutase treatment to prevent apoptosis of single cells. Additionally, cultures of stem cells can often spontaneously differentiate, particularly after overgrowth or stressful procedures. Isolated loci of differentiated cells may be removed directly by aspirating areas of the well, and this is particularly effective during EDTA splitting. For highly differentiated cultures, however, several passages are often required to regain a healthy pluripotent population. Plating at high density following EDTA splitting (1:3 ratio) can promote iPSCs to outcompete differentiated cells. Alternatively, a modified version of the EDTA split is also included below to remove spontaneously differentiated cells, which takes advantage of higher adhesion of differentiated cells compared to iPSCs.
EDTAFrom 18 to 21 steps
Aspirate culture medium and rinse with one-half culture volume of PBS.
Note
Since EDTA works by chelating the calcium ions necessary for iPSC attachment, be sure the PBS does not contain calcium.
Aspirate PBS and add one-half culture volume of EDTA solution (0.5 millimolar (mM) in PBS).
Incubate for 00:05:00 to 00:10:00 at Room temperature .
Note
Exact timing varies by cell line, but 7 min is typical for hiPSCs. Cell colonies should be opaque to the naked eye, and colony edges should be just starting to detach when viewed under a microscope. Single-cell dissociation may be achieved by incubating for up to 15 min, and time may be reduced by incubating plates at 37 °C .
If colonies begin sloughing off, collect EDTA and cells in a conical tube, rinse with culture medium and add to the tube, centrifuge 00:05:00 at 200 x g , aspirate supernatant, resuspend in culture medium supplemented with 10 micromolar (µM) ROCK inhibitor, and plate cells.
Reduce EDTA incubation time in future passages.
While incubating, prepare recipient Matrigel-coated plates by aspirating Matrigel solution and loading with three-quarters volume of culture medium.
Note
For example, 1.5 mL medium/well of a 6-well dish. Supplementation of 5 micromolar (µM) ROCK inhibitor is optional to improve survival.
Aspirate EDTA solution, taking care not to disturb cell colonies, which should remain attached.
Note
Small-scale differentiation may be removed at this step by directly aspirating areas of the well which have noticeable bumps (particularly in themiddle of large colonies) or by designating areas for removal beforehand with an object marker microscope objective.
Dissociate cells by pipetting two to three times with culture medium equivalent to half of the denominator of the splitting ratio.
Note
Typical splitting ratios for 6-well plates are between 1:6 and 1:12, for which 3 and 6 ml medium should be used, respectively. Mix well, but avoid pipetting up and down more than three times in order to keep cell clumps intact. If colonies remain attached, dispense medium, gently scrape the bottom of the well with the end of the pipet, and pipet up and down twice to mix. Increase EDTA incubation time in future passages.
Add one-fourth of the volume of culture medium with cells to each recipient well, and discard any excess cells.
Return plate to 37 °C incubator and evenly distribute cells by gently shaking platefront-to-back and side-to-side.
Freezing
Freezing
Note
Culturing over many passages may result in mutations and genetic drift, and stressful events can select for abnormal genotypes such as oncogene mutations or chromosomal deletions and rearrangements (Merkle et al., 2017). To circumvent these adverse outcomes and to provide backup in case of contamination, several vials of cells should be cryopreserved immediately after cell line isolation. Additionally, freezing cell clones during validation reduces reagent use, and expanding cultures and freezing a large, pooled batch of cells on the same date with the same passage number provides useful downstream reproducibility.
Prepare cells as for an EDTA split (see above). During EDTA incubation, label cryovials and formulate cryopreservation medium by combining culture medium with 10 % DMSO.
Note
Optionally, the addition of 20 % fetal bovine serum or knockout serum replacement to cryopreservation medium may improve viability. 90 % fetal bovine serum/10 % DMSO solutions (i.e., no culture medium) have also been used successfully. Note that if doxycycline-inducible promoters are integrated into the iPSC lines, we recommend the use of validated, tetracycline-free FBS for such purposes (e.g., Tet System Approved FBS from Clontech). Cryopreservation medium may also be purchased commercially (e.g., CryoStor CS10, STEMCELL Technologies cat. no. 07930).
Aspirate EDTA and dissociate cells with cryopreservation medium.
Note
It is optional to dissociate with culture medium and count cells (Phelan & May, 2015) in order to ensure that standard cell numbers are frozen across samples, typically 1 × 106 cells/vial. This is followed by centrifugation, aspiration of culture medium, and resuspension in an appropriate volume of cryopreservation medium. For routine applications, however, 1 mL to 2 mL cryopreservation medium per well of a 6-well plate at approximately 80 % confluency provides adequate cell density for a healthy thaw. Steps 3 to 4 should be completed as quickly as possible to reduce DMSO exposure.
Transfer 1 mL cryopreservation cell suspension to each 1.5 ml cryovial and freeze in a CoolCell freezing container or Mr. Frosty isopropanol caddy at -80 °C for 02:00:00 to overnight.
Note
Gradually reducing temperature by 1°C/minute improves viability.
Transfer cryovials to liquid nitrogen for long-term storage.
Note
Cryovials should be placed on dry ice for transit from the freezer to the liquid nitrogen tank to prevent thawing.
Note
Manual changes in the composition of an iPSC population may be accomplished by either isolating a desired colony into a separate culture (manual passage, or pick-to-keep) or by scraping away undesired cells for aspiration (pick-to-remove). In practice, manual passaging is an important part of the clonal isolation protocol below, and picking-to-remove is most commonly used to remove isolated areas of spontaneously differentiated cells from maintenance cultures or to provide more room for the desired cells to grow prior to picking-to-keep. Proper sterile technique is essential during both of these procedures,
as plates may be uncovered in the biosafety cabinet for extended periods of time.
Pick-to-keep5 steps
Manual changes in the composition of an iPSC population accomplished by isolating a desired colony into a separate culture (manual passage, or pick-to-keep).
Prepare a recipient 24-well plate by aspirating Matrigel solution and adding 250 μl/well of culture medium supplemented with 10 micromolar (µM) Y-27632 ROCK inhibitor.
Place the cell culture dish on a picking microscope in a sterile enclosure.
Note
A microscope (e.g., Lumascope) connected to a screen or tablet computer may be sterilized and kept inside a BSC. Alternatively, a PCR enclosure with a stereoscope that can be sterilized by ethanol and/or UV treatment provides sufficient protection.
Remove the cover of the culture dish, center the colony desired to be picked, flush the P1000 tip with medium to avoid cell retention, and align the end of a P1000 pipet tip just above the colony.
Note
Colonies are often pre-selected with a marking objective, which leaves a 1.8 mm ring on the bottom of the culture dish. Colonies approximately the size of the inside of the ring are of ideal size for picking, and a wide-diameter tip is desirable to reduce shear forces on the cells. P200 or smaller tips should not be used. Set the pipet to 250 μl. Balance the tip on your opposite index finger to improve stability and control.
Lower the pipet tip until it makes contact with the culture surface.
Note
If necessary, raise the plunger of the pipettor slightly to remove the air bubble for better viewing.
Gently and slowly scrape the bottom of the well with the tip, slowly raise the plunger to detach cells in strips, and collect in the pipet tip.
Note
Keep a shallow angle with the plate and avoid pressing down on the plate. Raise the plunger more quickly to provide more force if cells remain stuck or are close to the well wall.
Deposit the picked cells (in 250 µL medium) into the destination well.
Note
If picking has been slow, cells may be stuck to the inside of the tip, so check the well under the microscope before disposing of the tip to ensure that cells are present. Try to avoid pipetting multiple times in the well in order to keep cells clumped. If the clone was not completely picked, the same tip may be reused to acquire more cells; otherwise, change pipet tips between each clone.