Oct 21, 2020

Public workspaceCell seeding V.2

This protocol is a draft, published without a DOI.
  • 1Nanyang Technological University
PMAT0001
Icon indicating open access to content
QR code linking to this content
Protocol Citation: PMAT0001 2020. Cell seeding. protocols.io https://protocols.io/view/cell-seeding-bnp8mdrwVersion created by PMAT0001
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: October 21, 2020
Last Modified: October 21, 2020
Protocol Integer ID: 43488
Materials
MATERIALS
Reagent1X Dulbecco’s Phosphate Buffered Saline (DPBS) Thermo Fisher ScientificCatalog #14190094
ReagentPenicillin StreptomycinInvitrogen - Thermo FisherCatalog #15140 122
ReagentDMEMInvitrogen - Thermo FisherCatalog #11885
ReagentFoetal Bovine Serum (FBS) Triple 0.1 µm Sterile Filtered 500 ml Australian OriginSeranaCatalog #FBS-AU-015
Before start
- Asceptic techniques***
- Wipe down hood and every item introduced into the hood with 70% ethanol
Take cells from the freezer and thaw for Duration00:02:00 in theTemperature37 °C water bath.

2m
Take a T75 plate and place all needed items in ethanol-sterilized laminar flow hood.
Draw the needed amount of cells into a 50mL centrifuge tube.
Add aboutAmount7 mL of PBS into the centrifuge tube.

Spin the mixture in a centrifuge for Duration00:03:00 at 1500rpm.

3m
Decant supernatant and add PBS to the cell pellet once again.
Resuspend cell pellet with the PBS using a micropipette.
Repeat steps 5 to 7.
Add DMEM (with FBS and antibiotics) into centrifuge tube and resuspend cell pellet.
Add DMEM to the T75 plate (sufficient to cover the entire surface area, about Amount5 mL ).

Add Amount5 mL of the cell mixture into the T75 flask.

Incuabte T75 flask atTemperature37 °C , 5% CO2.