# hiPSC Culture Discussion **URL:** https://forum.allencell.org/c/hipsc-culture/10.md [Latest](https://forum.allencell.org/latest.md) · [Categories](https://forum.allencell.org/categories.md) · [Tags](https://forum.allencell.org/tags.md) --- ## [About the hiPSC Culture Discussion category](https://forum.allencell.org/t/about-the-hipsc-culture-discussion-category/24)
**Author:** [@cells](https://forum.allencell.org/u/cells)\ **Replies:** 0
This forum allows users to discuss culture techniques, tips and issues that arise when thawing, culturing, maintaining and differentiating human iPSCs. --- ## [Western Blot background GFP bands-- how did you reduce this in WTC-11?](https://forum.allencell.org/t/western-blot-background-gfp-bands-how-did-you-reduce-this-in-wtc-11/796)
**Author:** [@MaggietheFuqua](https://forum.allencell.org/u/MaggietheFuqua)\ **Replies:** 1\ **Last updated:** [April 29, 2026, 5:12pm UTC](https://forum.allencell.org/t/western-blot-background-gfp-bands-how-did-you-reduce-this-in-wtc-11/796 "2026-04-29T17:12:32Z")
Hi there, From your Roberts et al., 2017 pub, your GFP Western Blots look very clean, but I’m using the same antibody specified and getting so many background bands in both WT and my mEGFP-tagged line lysates. Any chanc… --- ## [Pictures of Healthy vs Unhealthy hiPSC colonies](https://forum.allencell.org/t/pictures-of-healthy-vs-unhealthy-hipsc-colonies/26)
**Author:** [@cells](https://forum.allencell.org/u/cells)\ **Replies:** 5\ **Last updated:** [January 20, 2026, 11:03pm UTC](https://forum.allencell.org/t/pictures-of-healthy-vs-unhealthy-hipsc-colonies/26 "2026-01-20T23:03:54Z")
How should healthy iPSC colonies look? What characteristics define a bad colony? --- ## [iPSC line AICS-0090-391 adipogenic differentiation](https://forum.allencell.org/t/ipsc-line-aics-0090-391-adipogenic-differentiation/785)
**Author:** [@Carolinepancera](https://forum.allencell.org/u/Carolinepancera)\ **Replies:** 0\ **Last updated:** [November 19, 2025, 10:00pm UTC](https://forum.allencell.org/t/ipsc-line-aics-0090-391-adipogenic-differentiation/785 "2025-11-19T22:00:38Z")
I am currently planning experiments involving the iPSC line AICS-0090-391 (WTC-CLYBL-dCas9-TagBFP-KRAB-cl391). I would like to know whether this specific line has been previously used or validated for adipogenic differe… --- ## [Thaw, expand and maintain MONO-ALLELIC mEGFP-TAGGED SOX2 cells in E8+ flex medium (Invitrogen) on Vitronectin-N coated wells](https://forum.allencell.org/t/thaw-expand-and-maintain-mono-allelic-megfp-tagged-sox2-cells-in-e8-flex-medium-invitrogen-on-vitronectin-n-coated-wells/771)
**Author:** [@leahpeng](https://forum.allencell.org/u/leahpeng)\ **Replies:** 1\ **Last updated:** [July 30, 2025, 8:09pm UTC](https://forum.allencell.org/t/thaw-expand-and-maintain-mono-allelic-megfp-tagged-sox2-cells-in-e8-flex-medium-invitrogen-on-vitronectin-n-coated-wells/771 "2025-07-30T20:09:44Z")
Hi, We would like to thaw, expand and maintain MONO-ALLELIC mEGFP-TAGGED SOX2 cells in E8+ flex medium (Invitrogen) on Vitronectin-N coated wells, which is different method comparing to the protocol provided by the Cori… --- ## [Tips for reducing non-specific antibody binding in WTC-11 cells on coated glass](https://forum.allencell.org/t/tips-for-reducing-non-specific-antibody-binding-in-wtc-11-cells-on-coated-glass/761)
**Author:** [@MaggietheFuqua](https://forum.allencell.org/u/MaggietheFuqua)\ **Replies:** 0\ **Last updated:** [April 23, 2025, 7:54pm UTC](https://forum.allencell.org/t/tips-for-reducing-non-specific-antibody-binding-in-wtc-11-cells-on-coated-glass/761 "2025-04-23T19:54:02Z")
Hello! I’ve been optimizing antibodies for IF using the WTC-11 cells and have utilized both Matrigel and Vitronectin, but am still seeing non-specific binding in my secondary-only controls in both cell-containing and ce… --- ## [Culturing hiPSC and Other Cell Lines in Same Lab Space](https://forum.allencell.org/t/culturing-hipsc-and-other-cell-lines-in-same-lab-space/760)
**Author:** [@drlatham77](https://forum.allencell.org/u/drlatham77)\ **Replies:** 1\ **Last updated:** [April 2, 2025, 8:51pm UTC](https://forum.allencell.org/t/culturing-hipsc-and-other-cell-lines-in-same-lab-space/760 "2025-04-02T20:51:22Z")
Hello! Our lab has a single small space for culturing cells. We have been using the Allen ihPSC cells and are looking into bringing in another cell line, 3T3. I know that HeLa cells have issues with ‘jumping’ between cu… --- ## [AICS-0126-068:WTC-mEGFP-CDH5 and AICS-0126-041:WTC-mEGFP-CDH5](https://forum.allencell.org/t/aics-0126-068-wtc-megfp-cdh5-and-aics-0126-041-wtc-megfp-cdh5/758)
**Author:** [@Tatjana](https://forum.allencell.org/u/Tatjana)\ **Replies:** 1\ **Last updated:** [March 20, 2025, 8:26pm UTC](https://forum.allencell.org/t/aics-0126-068-wtc-megfp-cdh5-and-aics-0126-041-wtc-megfp-cdh5/758 "2025-03-20T20:26:22Z")
Hi Allen team, We are thinking to purchase a VE-cadherin tagged iPSC line and would like to ask you whether the monoallelic (AICS-0126-068) or biallelic (AICS-0126-041) one would be the most favorable to visualize the e… --- ## [Protocol for Parental Cell Line (GM25256)](https://forum.allencell.org/t/protocol-for-parental-cell-line-gm25256/748)
**Author:** [@llachowski](https://forum.allencell.org/u/llachowski)\ **Replies:** 1\ **Last updated:** [February 17, 2025, 10:12pm UTC](https://forum.allencell.org/t/protocol-for-parental-cell-line-gm25256/748 "2025-02-17T22:12:38Z")
The protocol you provide for your cell lines (GM25256\*l) is slightly different from the protocol provided for the parental line (GM2526). My lab is interested in purchasing and culturing both your cell lines and the pare… --- ## [AICS-0037-172-MONO-ALLELIC mEGFP-TAGGED TNNI iPSCs RFP Signal](https://forum.allencell.org/t/aics-0037-172-mono-allelic-megfp-tagged-tnni-ipscs-rfp-signal/742)
**Author:** [@Mani1830](https://forum.allencell.org/u/Mani1830)\ **Replies:** 1\ **Last updated:** [February 4, 2025, 1:06am UTC](https://forum.allencell.org/t/aics-0037-172-mono-allelic-megfp-tagged-tnni-ipscs-rfp-signal/742 "2025-02-04T01:06:18Z")
Hi team, We differentiated the hiPSCs into cardiomyocytes and we observed that the RFP was observed but not GFP expression. Moreover, we didn’t observe the beating of the cardiomyocytes. Any suggestion would be highly a… --- ## [Advice of Lipofectamine STEM protocol](https://forum.allencell.org/t/advice-of-lipofectamine-stem-protocol/735)
**Author:** [@AbhiRBio](https://forum.allencell.org/u/AbhiRBio)\ **Replies:** 1\ **Last updated:** [December 4, 2024, 5:27pm UTC](https://forum.allencell.org/t/advice-of-lipofectamine-stem-protocol/735 "2024-12-04T17:27:23Z")
Hi! I recently tried to transfect WTC10 stem cells with a gene under the EF1a promoter. ~30k cells were seeded per well of a CellVisC4-1.5H-N chambered slide coated with vitronectin, in mTeSR+ medium with 1X RevitaCell … --- ## [How to keep cardiac differentiated cells from coming apart lifting from well](https://forum.allencell.org/t/how-to-keep-cardiac-differentiated-cells-from-coming-apart-lifting-from-well/724)
**Author:** [@hartw006](https://forum.allencell.org/u/hartw006)\ **Replies:** 1\ **Last updated:** [July 30, 2024, 4:18pm UTC](https://forum.allencell.org/t/how-to-keep-cardiac-differentiated-cells-from-coming-apart-lifting-from-well/724 "2024-07-30T16:18:19Z")
How do you to keep cardiac differentiated cells from coming apart and lifting from well? My cells beat so hard they do not last that long, a month a most. --- ## [Cardiomyocyte Differentiation Issue](https://forum.allencell.org/t/cardiomyocyte-differentiation-issue/99)
**Author:** [@hwidyastuti](https://forum.allencell.org/u/hwidyastuti)\ **Replies:** 12\ **Last updated:** [February 22, 2024, 2:27pm UTC](https://forum.allencell.org/t/cardiomyocyte-differentiation-issue/99 "2024-02-22T14:27:08Z")
Hi, I’m just starting the Cardiomyocyte differentiation using the Allen Institute protocol. Currently at day 8 of differentiation and I’m starting to see some isolated beating clusters. I noticed that in the protocol the… --- ## [iPS cardiac differentiation with lactate](https://forum.allencell.org/t/ips-cardiac-differentiation-with-lactate/664)
**Author:** [@hartw006](https://forum.allencell.org/u/hartw006)\ **Replies:** 1\ **Last updated:** [November 1, 2023, 9:26pm UTC](https://forum.allencell.org/t/ips-cardiac-differentiation-with-lactate/664 "2023-11-01T21:26:19Z")
Hi, Can you suggest a protocol for lactate purification after wnt-modulated differentiation? I have been unable to find a detailed protocol. thanks, Lynn --- ## [Reason for Matrigel Concentration Recommended](https://forum.allencell.org/t/reason-for-matrigel-concentration-recommended/650)
**Author:** [@guohaodailab](https://forum.allencell.org/u/guohaodailab)\ **Replies:** 1\ **Last updated:** [August 16, 2023, 6:06pm UTC](https://forum.allencell.org/t/reason-for-matrigel-concentration-recommended/650 "2023-08-16T18:06:09Z")
Hi, we received some cells recently from your cell catalog and are in the process of thawing them. The matrigel concentration recommended (0.337 mg/mL) is a few fold higher than what is instructed by the manufacturer (C… --- ## [Defrosting in 100mm dish](https://forum.allencell.org/t/defrosting-in-100mm-dish/651)
**Author:** [@guohaodailab](https://forum.allencell.org/u/guohaodailab)\ **Replies:** 1\ **Last updated:** [August 16, 2023, 6:00pm UTC](https://forum.allencell.org/t/defrosting-in-100mm-dish/651 "2023-08-16T18:00:36Z")
Hi, We recently received cells from your catalog and noticed the directions to thaw into a 100mm dish specifically and to NOT thaw into a 6 well plate. Can you explain the decision behind this? As the cells are so valu… --- ## [Code for counting individual cells in colonies](https://forum.allencell.org/t/code-for-counting-individual-cells-in-colonies/644)
**Author:** [@guohaodailab](https://forum.allencell.org/u/guohaodailab)\ **Replies:** 1\ **Last updated:** [July 18, 2023, 6:06pm UTC](https://forum.allencell.org/t/code-for-counting-individual-cells-in-colonies/644 "2023-07-18T18:06:35Z")
Wondering if there is any available code or macro for counting individual cells in hPSC colonies? There was a method mentioned at ISSCR conference on how to get an accurate single cell count from colonies. Thanks! Stef… --- ## [Weekend free protocols for iPS maintenance](https://forum.allencell.org/t/weekend-free-protocols-for-ips-maintenance/586)
**Author:** [@hperera](https://forum.allencell.org/u/hperera)\ **Replies:** 3\ **Last updated:** [April 20, 2023, 5:37pm UTC](https://forum.allencell.org/t/weekend-free-protocols-for-ips-maintenance/586 "2023-04-20T17:37:56Z")
Can I passage cells on a Thursday or a Friday and do a double feed on a Saturday so that I can skip a feed on a Sunday? --- ## [Antibiotic resistance in WTC derived lines?](https://forum.allencell.org/t/antibiotic-resistance-in-wtc-derived-lines/614)
**Author:** [@hartw006](https://forum.allencell.org/u/hartw006)\ **Replies:** 1\ **Last updated:** [March 9, 2023, 11:42pm UTC](https://forum.allencell.org/t/antibiotic-resistance-in-wtc-derived-lines/614 "2023-03-09T23:42:40Z")
Do any of the cell lines have antibiotic resistance genes? I am interested in using Puromycin selection --- ## [Cryo-preserving in CryoStor?](https://forum.allencell.org/t/cryo-preserving-in-cryostor/596)
**Author:** [@OBEID](https://forum.allencell.org/u/OBEID)\ **Replies:** 2\ **Last updated:** [January 20, 2023, 4:00am UTC](https://forum.allencell.org/t/cryo-preserving-in-cryostor/596 "2023-01-20T04:00:25Z")
I would like to know if i can cryo-preserve my cell line (AISC-0012) in CryoStorCS10 from StemCell Technologies (#07959). Thank you Pat --- ## [Dilution of Matrigel : which media?](https://forum.allencell.org/t/dilution-of-matrigel-which-media/595)
**Author:** [@OBEID](https://forum.allencell.org/u/OBEID)\ **Replies:** 2\ **Last updated:** [January 19, 2023, 5:10am UTC](https://forum.allencell.org/t/dilution-of-matrigel-which-media/595 "2023-01-19T05:10:07Z")
I have a doubt… In the protocol version 1.7, I read that you have to dilute the Matrigel in DMEM/F12 to make the coating. But in paragraph 13 of the Passaging section, I understand that the Matrigel is prepared in mTeSR… --- ## [Can I use Glutamax versions of media and still get good differentiation to cardiac?](https://forum.allencell.org/t/can-i-use-glutamax-versions-of-media-and-still-get-good-differentiation-to-cardiac/580)
**Author:** [@hartw006](https://forum.allencell.org/u/hartw006)\ **Replies:** 1\ **Last updated:** [January 5, 2023, 6:55pm UTC](https://forum.allencell.org/t/can-i-use-glutamax-versions-of-media-and-still-get-good-differentiation-to-cardiac/580 "2023-01-05T18:55:07Z")
Can I use RPMI with glutamax (also other versions with glutamine replaced with di peptide- Ala-Gln) and still have good differentiation to cardiac? I assume that mTesr1 Plus is similarly a replacement of glutamax for g… --- ## [Conflicting Matrigel instructions](https://forum.allencell.org/t/conflicting-matrigel-instructions/549)
**Author:** [@en2b](https://forum.allencell.org/u/en2b)\ **Replies:** 1\ **Last updated:** [November 10, 2022, 7:51pm UTC](https://forum.allencell.org/t/conflicting-matrigel-instructions/549 "2022-11-10T19:51:07Z")
I recently received a new vial of cells and the single sheet titled “Allen Cell Collection: Getting Started” states to coat TC plates with Matrigel diluted 1:30. However, the Culture and Freezing Methods for WTC Derived … --- ## [Matrigel vs geltrex](https://forum.allencell.org/t/matrigel-vs-geltrex/449)
**Author:** [@Tiam](https://forum.allencell.org/u/Tiam)\ **Replies:** 3\ **Last updated:** [November 10, 2022, 4:34pm UTC](https://forum.allencell.org/t/matrigel-vs-geltrex/449 "2022-11-10T16:34:48Z")
Hi, is it possible to replace matrigel with geltrex? --- ## [Follow up on earlier topic - GFR vs. hESC qualified matrigel](https://forum.allencell.org/t/follow-up-on-earlier-topic-gfr-vs-hesc-qualified-matrigel/545)
**Author:** [@ssasi](https://forum.allencell.org/u/ssasi)\ **Replies:** 0\ **Last updated:** [November 7, 2022, 1:38pm UTC](https://forum.allencell.org/t/follow-up-on-earlier-topic-gfr-vs-hesc-qualified-matrigel/545 "2022-11-07T13:38:46Z")
This is a follow up on an earlier thread here (04/2019). Please see below - We’ve had questions recently about the type of Matrigel to use with the Allen Institute for Cell Science cell lines. To our knowledge… witho… --- ## [Types of Matrigel- GFR? hESC qualified?](https://forum.allencell.org/t/types-of-matrigel-gfr-hesc-qualified/90)
**Author:** [@amandah](https://forum.allencell.org/u/amandah)\ **Replies:** 1\ **Last updated:** [November 4, 2022, 7:53pm UTC](https://forum.allencell.org/t/types-of-matrigel-gfr-hesc-qualified/90 "2022-11-04T19:53:52Z")
We’ve had questions recently about the type of Matrigel to use with the Allen Institute for Cell Science cell lines. To our knowledge… without doing lot testing in your own lab, the “safest” bet would be to use an hESC… --- ## [Choice of lines for monitoring cardiac differentiation](https://forum.allencell.org/t/choice-of-lines-for-monitoring-cardiac-differentiation/514)
**Author:** [@mwcosta](https://forum.allencell.org/u/mwcosta)\ **Replies:** 1\ **Last updated:** [September 22, 2022, 10:31pm UTC](https://forum.allencell.org/t/choice-of-lines-for-monitoring-cardiac-differentiation/514 "2022-09-22T22:31:06Z")
Hi, We are are structuring some experiments to test efficiency of transfection during iPSC-CM differentiation stages and would like to use some line that expresses cardiac markers to facilitate the analysis. We are curr… --- ## [mGFP-Tagged MYL2](https://forum.allencell.org/t/mgfp-tagged-myl2/370)
**Author:** [@hxt132](https://forum.allencell.org/u/hxt132)\ **Replies:** 1\ **Last updated:** [November 17, 2021, 11:00pm UTC](https://forum.allencell.org/t/mgfp-tagged-myl2/370 "2021-11-17T23:00:40Z")
When will this hipsc cell line (AICS-0060-027) show mGFP signal? Will it appear mGFP signal before cardiac induction? --- ## [hiPSC differentiation](https://forum.allencell.org/t/hipsc-differentiation/342)
**Author:** [@dinunzio](https://forum.allencell.org/u/dinunzio)\ **Replies:** 1\ **Last updated:** [August 6, 2021, 1:46pm UTC](https://forum.allencell.org/t/hipsc-differentiation/342 "2021-08-06T13:46:54Z")
Can you advice a good protocol for differentiation of your hiPSC in macrophages and/or in CD4+T cells? --- ## [HLA type of WTC AKA WTC11](https://forum.allencell.org/t/hla-type-of-wtc-aka-wtc11/333)
**Author:** [@dkoelle](https://forum.allencell.org/u/dkoelle)\ **Replies:** 1\ **Last updated:** [June 2, 2021, 10:29pm UTC](https://forum.allencell.org/t/hla-type-of-wtc-aka-wtc11/333 "2021-06-02T22:29:58Z")
Does anyone know the HLA type of these cells? Thanks [Next page](https://forum.allencell.org/c/hipsc-culture/10.md?page=1)