Difference between revisions of "Haynes:MamCultureMedia"

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("Wild type" stock cells (non-transgenic))
 
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| '''Cell line''' || '''Incomplete media''' || '''Supplements for complete media (required)'''
 
| '''Cell line''' || '''Incomplete media''' || '''Supplements for complete media (required)'''
 
|-
 
|-
| HEK293 || DMEM high glucose || 10% FBS, 1% pen/strep
+
| HEK293 (adherent) || DMEM, high glucose || 10% FBS, 1% pen/strep
 
|-
 
|-
| K-562 (suspension) || Iscove's Modified Dulbecco's Medium (IMDM) || 10% FBS, 1% pen/strep
+
| [http://atcc.org/Products/All/CCL-243.aspx#culturemethod K-562] (suspension) || Iscove's Modified Dulbecco's Medium (IMDM) || 10% FBS, 1% pen/strep
 
|-
 
|-
| SK-N-SH (adherent) || Eagle's Minimum Essential Medium (EMEM) || 10% FBS, 1% pen/strep
+
| [http://atcc.org/Products/All/HTB-11.aspx#culturemethod SK-N-SH] (adherent) || Eagle's Minimum Essential Medium (EMEM) || 10% FBS, 1% pen/strep
 
|-
 
|-
| U2OS (adherent) || McCoy's 5A || 10% FBS, 1% pen/strep  
+
| [http://atcc.org/Products/All/HTB-96.aspx#culturemethod U-2 OS] (adherent) || McCoy's 5A || 10% FBS, 1% pen/strep
 +
|-
 +
| [http://atcc.org/Products/All/HTB-20.aspx#culturemethod BT-474] (adherent<sup>1</sup>) || ATCC Hybri-Care Medium<sup>2</sup> || 10% FBS, 1% pen/strep, 1.5 g/L sodium bicarbonate
 +
|-
 +
| [http://atcc.org/Products/All/HTB-122.aspx#culturemethod BT-549] (adherent) || ATCC RPMI-1640 Medium || 10% FBS, 1% pen/strep, 0.0008 mg/mL human recombinant insulin
 +
|-
 +
| [http://atcc.org/Products/All/HTB-22.aspx#culturemethod MCF7] (adherent) || Eagle's Minimum Essential Medium (EMEM) || 10% FBS, 1% pen/strep, 0.01 mg/mL human recombinant insulin
 +
|-
 +
| [http://atcc.org/Products/All/CRL-10317.aspx#culturemethod MCF 10A] (adherent) || [http://bio.lonza.com/go/literature/356 MEGM BulletKit] <u>without</u> GA-1000 || 100 ng/mL cholera toxin<sup>3</sup>
 +
|-
 +
| [http://www.atcc.org/products/all/HTB-38.aspx HT-29] (adherent) || McCoy's 5A || 10% FBS, 1% pen/strep
 +
|}
 +
 
 +
 
 +
<sup>1</sup> BT-474 cells form adherent patches of epithelial-like cells. The patches are compact multilayered colonies that rarely become confluent.<br>
 +
<sup>2</sup> Hybri-Care Medium is a powder that needs to be dissolved in water before use.<br>
 +
<sup>3</sup> For best results, do not filter MCF 10A complete medium before use.
 +
 
 +
<br>
 +
 
 +
==Transgenic Cell Selection==
 +
{| class="wikitable" cellspacing=5px
 +
|- valign="top"
 +
| '''Cell Type''' || '''Genotype''' || '''Transfected Plasmid''' || '''Medium''' || '''Supplements<br>(required)''' || '''Selection<br>antibiotic(s)'''
 +
|-
 +
| U2OS (adherent) || Flp-in T-REx || pcDNA5/FRT, or V0200 || McCoy's 5A || 10% FBS-tf, 1 % pen/strep || 15 ug/mL blasticidin, 200 μg/mL hygromycin
 +
|-
 +
| U2OS (adherent) || misc. || MV2 or pcDNA3.1/puro || McCoy's 5A || 10% FBS-tf, 1 % pen/strep || 15 ug/mL blasticidin, 1.0 μg/mL puromycin
 
|}
 
|}
  
 
<br>
 
<br>
  
==Transgenic Cells==
+
==Transgenic Cell Maintenance==
 
{| class="wikitable" cellspacing=5px
 
{| class="wikitable" cellspacing=5px
 
|- valign="top"  
 
|- valign="top"  
 
| '''Cell Type''' || '''Genotype''' || '''Medium''' || '''Supplements<br>(required)''' || '''Maintenance<br>antibiotic(s)'''
 
| '''Cell Type''' || '''Genotype''' || '''Medium''' || '''Supplements<br>(required)''' || '''Maintenance<br>antibiotic(s)'''
 
|-
 
|-
| U2OS (adherent) || Flp-in T-REx || McCoy's 5A || 10% FBS, 1 % pen/strep || ## ug/mL puromycin
+
| U2OS (adherent) || Flp-in T-REx, untransfected || McCoy's 5A || 10% FBS-tf, 1 % pen/strep || 100 ug/mL Zeocin, 15 ug/mL blasticidin
 +
|-
 +
| U2OS (adherent) || Flp-in T-REx, hygro-transfected || McCoy's 5A || 10% FBS-tf, 1 % pen/strep || 15 ug/mL blasticidin, 60 μg/mL hygromycin
 
|-
 
|-
| HEK 293 (adherent) || Flp-in T-REx || DMEM || 10% FBS, 1 % pen/strep || ## ug/mL puromycin
+
| U2OS (adherent) || Flp-in T-REx, puro-transfected || McCoy's 5A || 10% FBS-tf, 1 % pen/strep || 15 ug/mL blasticidin, 0.5 μg/mL puromycin
 +
|-
 +
| HEK 293 (adherent) || Flp-in T-REx || DMEM, high glucose || 10% FBS-tf, 1 % pen/strep || 100 ug/mL Zeocin, 15 ug/mL blasticidin
 +
|-
 +
| HEK 293 (adherent) || luc Gal4-EED "23;4;9" (K. Hansen) || DMEM, high glucose || 10% FBS-tf, 1 % pen/strep || 0.5 ug/mL puromycin
 
|}
 
|}
  
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* FBS-tf = tetracycline-free FBS (required to maintain the "off" state of dox-inducible transgenes)
 
* FBS-tf = tetracycline-free FBS (required to maintain the "off" state of dox-inducible transgenes)
 
* pen/strep = penicillin and streptomycin anti fungal supplement
 
* pen/strep = penicillin and streptomycin anti fungal supplement
* Filter sterilize all media before use
+
* Filter-sterilize all media before use
 
* Store all media at 4°C
 
* Store all media at 4°C
  
  
 
</div>
 
</div>
 +
==BT-549==
 +
 +
Confluent T-75 flask of BT-549 cells, density ~430,000 cells/mL
 +
 +
[[Image:11-23-2015_BT549_confluent.tif | 500px]]

Latest revision as of 13:15, 20 April 2016

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Mammalian Culture Media

"Wild type" stock cells (non-transgenic)

Cell line Incomplete media Supplements for complete media (required)
HEK293 (adherent) DMEM, high glucose 10% FBS, 1% pen/strep
K-562 (suspension) Iscove's Modified Dulbecco's Medium (IMDM) 10% FBS, 1% pen/strep
SK-N-SH (adherent) Eagle's Minimum Essential Medium (EMEM) 10% FBS, 1% pen/strep
U-2 OS (adherent) McCoy's 5A 10% FBS, 1% pen/strep
BT-474 (adherent1) ATCC Hybri-Care Medium2 10% FBS, 1% pen/strep, 1.5 g/L sodium bicarbonate
BT-549 (adherent) ATCC RPMI-1640 Medium 10% FBS, 1% pen/strep, 0.0008 mg/mL human recombinant insulin
MCF7 (adherent) Eagle's Minimum Essential Medium (EMEM) 10% FBS, 1% pen/strep, 0.01 mg/mL human recombinant insulin
MCF 10A (adherent) MEGM BulletKit without GA-1000 100 ng/mL cholera toxin3
HT-29 (adherent) McCoy's 5A 10% FBS, 1% pen/strep


1 BT-474 cells form adherent patches of epithelial-like cells. The patches are compact multilayered colonies that rarely become confluent.
2 Hybri-Care Medium is a powder that needs to be dissolved in water before use.
3 For best results, do not filter MCF 10A complete medium before use.


Transgenic Cell Selection

Cell Type Genotype Transfected Plasmid Medium Supplements
(required)
Selection
antibiotic(s)
U2OS (adherent) Flp-in T-REx pcDNA5/FRT, or V0200 McCoy's 5A 10% FBS-tf, 1 % pen/strep 15 ug/mL blasticidin, 200 μg/mL hygromycin
U2OS (adherent) misc. MV2 or pcDNA3.1/puro McCoy's 5A 10% FBS-tf, 1 % pen/strep 15 ug/mL blasticidin, 1.0 μg/mL puromycin


Transgenic Cell Maintenance

Cell Type Genotype Medium Supplements
(required)
Maintenance
antibiotic(s)
U2OS (adherent) Flp-in T-REx, untransfected McCoy's 5A 10% FBS-tf, 1 % pen/strep 100 ug/mL Zeocin, 15 ug/mL blasticidin
U2OS (adherent) Flp-in T-REx, hygro-transfected McCoy's 5A 10% FBS-tf, 1 % pen/strep 15 ug/mL blasticidin, 60 μg/mL hygromycin
U2OS (adherent) Flp-in T-REx, puro-transfected McCoy's 5A 10% FBS-tf, 1 % pen/strep 15 ug/mL blasticidin, 0.5 μg/mL puromycin
HEK 293 (adherent) Flp-in T-REx DMEM, high glucose 10% FBS-tf, 1 % pen/strep 100 ug/mL Zeocin, 15 ug/mL blasticidin
HEK 293 (adherent) luc Gal4-EED "23;4;9" (K. Hansen) DMEM, high glucose 10% FBS-tf, 1 % pen/strep 0.5 ug/mL puromycin


Notes

  • FBS = fetal bovine serum
  • FBS-tf = tetracycline-free FBS (required to maintain the "off" state of dox-inducible transgenes)
  • pen/strep = penicillin and streptomycin anti fungal supplement
  • Filter-sterilize all media before use
  • Store all media at 4°C


BT-549

Confluent T-75 flask of BT-549 cells, density ~430,000 cells/mL

File:11-23-2015 BT549 confluent.tif