Stem Cells

High Performance Tlc Analysis And Immunostaining Protocol

High-Performance TLC Analysis and Immunostaining Protocol

Mammosphere Assay Of Mcf 7 Cells

Mammosphere Assay of MCF-7 cells

High Performance Tlc Analysis And Immunostaining

High-Performance TLC Analysis and Immunostaining

Passaging And Colony Expansion Of Human Pluripotent Stem Cells Preparation Of Imef Culture Medium

Several reagents must be prepared prior to maintaining human pluripotent stem cells (hPSCs) in culture. It is important to have a firm grasp of the characteristics of each reagent, including components, appropriate preparation, storage, and shelf life. The details of the preparation of preparation of iMEF culture medium

 

B2 Preparation of iMEF culture medium

 

Materials required

 

  • 5 ml sterile serological pipettes

 

  • 10 ml sterile serological pipettes

 

  • 25 ml sterile serological pipettes

 

  • 500 ml bottle connected with a 0. 22 μm Stericup

 

 

Reagents

 

  • DMEM-liquid (Invitrogen 11965-118)

 

  • MEM Non-Essential Amino Acid Solution (NEAA) (Invitrogen 11140-050)

 

  • Heat Inactivated Fetal Bovine Serum (HI-FBS) (Invitrogen 16000-069)

 

  • 70% ethanol (Diluted from 95% ethanol, Fisher NC9608803)

 

Procedure

 

Preparation of iMEF culture media

 

In the hood, open a 500 ml filter and bottle unit.

 

Label the bottle: iMEF and date of preparation, expiration date- 14 days after media preparation.

 

Add the appropriate amount of ingredients to the 500 ml filter cup as below:

 

          Ingredients           100 (ml)         500 (ml)

 

NEAA                             1                        5

Heat inactivated FBS      10                      50

DMEM                            90                      445

 

 

  • The DMEM may be measured by pouring directly into the graduated filter cup of the filter/bottle unit. The other solutions should be added using serological pipettes.

 

  • Scale up or down proportionally if another quantity of medium is needed.

 

  • Filter the media through the Stericup filter into the attached bottle.

 

  • Store the medium bottle at 4°C, and use the medium within 14 days.

Passaging And Colony Expansion Of Human Pluripotent Stem Cells B1 Reagent Preparation

Several reagents must be prepared prior to maintaining human pluripotent stem cells (hPSCs) in culture. It is important to have a firm grasp of the characteristics of each reagent, including components, appropriate preparation, storage, and shelf life. The details of the preparation of 0.1% gelatin solution

 

B1 Preparation of 0.1% gelatin solution

 

Materials Required

 

  • 500 ml sterile glass bottle

 

  • Weigh boat

 

Reagents

 

  • Gelatin, Porcine

 

  • Endotoxin-free

 

 

Method

 

  • Adjust the volume of water and gelatin powder proportionally if a volume other than 500 ml of gelatin is prepared.

 

  • Add 0.5 g of gelatin powder to a clean 500 ml pyrex bottle.

 

  • Add 500 ml endotoxin-free (e.g., MilliQ) water to the bottle.

 

  • Swirl to mix. The gelatin is not soluble at this stage,.

 

  • Autoclave for 30 minutes within 2 hours after mixing.

 

  • Cool the 0.1% gelatin solution to room temperature and store at 4–8°C until use. Use this solution within two months of preparation.