Subculturing Cells Protocol

Subculturing Cells Protocol

Author:  Sterling Mikkelson

Date Revised: 12/14/11

Cell Line(s):  B16F10, 2H11, FSaII, MDCKII, A549, HUVEC**, Fibroblast, HepG2*

Complete Media (Sterile Filtered): 

RPMI 1640 – 15% Fetal Bovine Serum (FBS) + 1% Penicillin/Streptomycin

**HUVEC – 20% FBS+ 1% Penicillin/Streptomycin

                        *EMEM – 10% FBS – 1% Penicillin/Streptomycin

Materials:

·         Pipette Controller

·         Confluent (80%) cell flask

·         1 mL aspirating pipette

·         Hank’s Balanced Salt Solution (HBSS)

·         5 mL pipette

·         0.25% Trypsin

·         0.05% Trypsin (HUVEC)

·         Complete Media

·         10 mL pipette

·         15 mL conical tube

·         Centrifuge (1500 rpm)

·         P1000 Pipettor

Procedure:

1.    Hold T-150 flask upright and using a 1mL aspirating pipette connected to vacuum line, aspirate media from flask

2.    Add 10 mL HBSS to rinse cells by gently rocking in a circular motion

3.    Remove HBSS by aspiration

4.    Add 5 mL of Trysin/EDTA and incubate at 37°C for 5-10min or until 90% of cells have become detached.

5.    Remove flask from incubator and add 6 mL of appropriate media to the cells containing trypsin.

6.    Collect cell solution with 10 mL pipette, and transfer to 15 mL conical tube.

7.    Cap and centrifuge for 5 minutes at 1500 rpm.

8.    Aspirate supernatant and add 3-5 mL fresh media.  Use gentle pipetting to break cell pellet.

9.    Count cells under a hemacytometer. (http://www.microbehunter.com/2010/06/27/the-hemocytometer-counting-chamber/)

10.  Add approximately 5x10⁵ cells from cell stock into T-150 flasks and add ≥15 mL complete media.

11.  Incubate flask at 37°C