MMG203 Mammalian Cell Culture

Schedule Spring 2005

Tuesday & Thursday

Lecture: 12:30 – 1:45PM Given C443

Lab: 2 – 4:45PM Stafford 104

 

WEEK 1

Tuesday January 18:

            LECTURE:

Course mechanics

Preparing a Cell Culture Laboratory

Cell culture contamination

 

            LABORATORY: 

Clean the hood, CO2 incubator, fridge and acquire materials

 

Thursday January 20:

            LECTURE:

Medium components and Preparation

Cell Culture Origins, Cell Types

Aseptic technique

 

            LABORATORY:        

Make DMEM medium, sterilize and aliquot

Make 90% DMEM/10% FBS, pH

                        Aliquot trypsin

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WEEK 2

Tuesday January 25:

            LECTURE:    

QUIZ

                        Subculturing Cells

                        Cell enumeration

                       

            LABORATORY:        

Obtain practice cells make visual observations

                        Harvest cells on bench

Determine cells/ml and total cells by hemocytometer counts with trypan blue

 

Thursday January 27:

            LECTURE:

                        HeLa & NIH3T3’s

                        Viability staining with Propidium Iodide

 

            LABORATORY:        

Obtain flasks of cells and harvest

                        Determine cells/ml by hemocytometer counts

At least 2 different people must do the counts and the final cells/ml will be an average of these readings

Viability analysis by FLOW cytometry

                        Make 4 T75 stock flasks

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WEEK 3

Sunday January 30:

            LABORATORY:

                        HeLa labs must trypsinize the stock flasks and allow to resettle

 

Tuesday February 1:

            LECTURE:

                        Phases of growth

                        Growth curves

 

            LABORATORY:        

Harvest 4 stock flasks, combine, and determine cells/ml

                        Seed 2 T75’s for stock flasks

                        Seed 9 T25’s for growth curve

                                    Three flasks at 3 x 105 cells/T25

Three flasks at 6 x 105 cells/T25

Three flasks at 1 x 106 cells/T25

 

Wednesday February 2:

            LABORATORY:      

Harvest 24 hr growth curve flasks – one each of the 3 x 105,

                                    6 x 105, and 1 x 106 flasks

For each time point 2 independent hemocytometer counts must be performed.  Determine cells/ml and total viable cells.

 

Thursday February 3:

            LECTURE:

                        QUIZ

                        Generation number, Multiplication rate, & Population doubling rate

 

            LABORATORY:        

Subculture stock flasks – prepare 3 T75’s

                        Harvest 48 hr growth curve flasks – one each of the 3 x 105,

                                    6 x 105, and 1 x 106 flasks.

For each time point 2 independent hemocytometer counts must be performed.  Determine cells/ml and total viable cells.

 

Friday February 4:

            LABORATORY:      

Harvest 72 hr growth curve flasks – one each of the 3 x 105,

                                    6 x 105, and 1 x 106 flasks.

For each time point 2 independent hemocytometer counts must be performed.  Determine cells/ml and total viable cells.

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WEEK 4

Sunday February 6:

            LABORATORY:

HeLa labs must trypsinize the stock flasks and allow to resettle

 

Tuesday February 8:

            LECTURE:

                        No lecture – go straight to lab

 

            LABORATORY:        

Subculture stock flasks

Prepare enough flasks to freeze down 10 aliquots of 1 x 106 cells/vial on Thursday

Review growth curve data, generate graphs, determine generation number, multiplication rate and population doubling time

 

Thursday February 10:

            LECTURE:

                        Cryopreservation of Cells & Thawing

 

            LABORATORY:        

                        Subculture stock flasks

                        Freeze cells at 1 x 106 cells/vial.  Do 10 vials.  Prepare freezing controls.

 

Friday February 11:

            LABORATORY:

                        Harvest the freezing control flasks and determine cells/ml and total cell numbers.

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WEEK 5

Sunday February 13:

            LABORATORY:

                        HeLa labs must trypsinize the stock flasks and allow to resettle

 

Tuesday February 15:

            LECTURE:

                        QUIZ

 

            LABORATORY:        

                        Subculture stock flasks

                        Thaw one vial from liquid nitrogen and plate into a T75

 

Wednesday February 16:

            LABORATORY:

                        Harvest the freezing control flask and determine cells/ml and total cell numbers.  Compare to other freezing controls to determine how the freezing protocol went.

 

Thursday February 17:

            LECTURE:

                        Mycoplasma

 

            LABORATORY:        

                        Subculture stock flasks

                        Prepare for formal presentation

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WEEK 6

Sunday February 20:

            LABORATORY:

                        HeLa labs must trypsinize the stock flasks and allow to resettle

 

Tuesday February 22:

            LECTURE: 

                        FORMAL PRESENTATIONS – Growth Curves, Freezing & Viability

 

            LABORATORY:

                        Subculture stock flask

                        Plate cells onto glass coverslips for mycoplasma protocol

                       

Thursday February 24:

            LECTURE:

                        QUIZ

                        Scientific Paper Discussion

 

            LABORATORY:        

                        Subculture stock flasks

                        Stain coverslips and visualize mycoplasma

 

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WEEK 7

Sunday February 27:

            LABORATORY:

                        HeLa labs must trypsinize the stock flasks and allow to resettle

                       

Tuesday March 1:

            NO CLASS: Town Meeting Day Recess

            LABORATORY:

                        Subculture stock flasks

 

Thursday March 3:

            LECTURE:

                        Introduction of exogenous DNA into mammalian cells

 

            LABORATORY:        

                        Subculture stock flasks

                        Seed T75 for harvest on Monday

                                    HeLa     5 x 105 cells/T75

                                    NIH3T3     3 x 105 cells/T75

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WEEK 8

Sunday March 6:

            LABORATORY:

                        HeLa labs must trypsinize the stock flasks and allow to resettle

 

Monday March 7:

            LABORATORY:      

                        Subculture T75 and seed three T25 flasks for transfection experiment 

                                    HeLa     6 x 105 cells/T25

                                    NIH3T3     7 x 105 cells/T25

 

Tuesday March 8:

            LECTURE:

                        Cell Cycle and FLOW analysis

                        Discuss 1997 Hanazono paper

 

            LABORATORY:        

                        Subculture stock flasks

                        Transfection of pEGFP-F with LipofectAMINE & PLUS

                        Medium must be changed 3-4 hrs after transfection

 

Wednesday March 9:

            LABORATORY:

                        Make visual observations of the transfection flasks for confluency and overall cell health.  Compare flasks 2 & 3 to the untransfected flask.

 

Thursday March 10:

            LECTURE:

                        QUIZ

                        siRNA

 

            LABORATORY:

                        Seed T75 for harvest on Monday

                                    HeLa     5 x 105 cells/T75

                                    NIH3T3     3 x 105 cells/T75

                        Cell Cycle analysis by FLOW of transfected cells

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WEEK 9

Monday March 14:

            LABORATORY:      

                        Seed cells for siRNA transfection in a 96-well plate

           

Tuesday March 15:

            LECTURE:

                        MID-TERM EXAM

 

            LABORATORY:        

                        siRNA Transfection

 

Thursday March 17:

            LECTURE: No lecture – begin lab immediately

 

            LABORATORY:

                        b-gal analysis of siRNA transfected cells

                        Discard any cells before going on Spring Break

 

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WEEK 10

Tuesday March 22 - Thursday March 24:

            SPRING BREAK – ENJOY!!!

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WEEK 11

Tuesday March 29:

            LECTURE:

                        QUIZ

                        C2C12 Differentiation & Muscle Physiology

 

            LABORATORY:        

                        Obtain T75’s of new cells and make observations of morphology

                        Make observations on cell morphology and confluency of C2C12’s in the T25 (this flask will be differentiated)

                        Prepare for formal presentations

 

 

Thursday March 31:

            LECTURE: 

                        FORMAL PRESENTATIONS – Mycoplasma, Cell Cycle & siRNA

 

            LABORATORY:        

                        Subculture cells in T75’s for harvest on Monday (see chart)

                        Switch C2C12 medium in the T25 to induce differentiation – DAY 1, make observations on cell morphology and confluency

 

Friday April 1:

            LABORATORY:      

                        Observe C2C12 culture – DAY 2.  Replace differentiation medium.

 

Saturday April 2:

            LABORATORY:

                        Observe C2C12 culture – DAY 3.  Replace differentiation medium.

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WEEK 12

Sunday April 3:

            LABORATORY:      

                        Observe C2C12 culture – DAY 4.  Replace differentiation medium.

 

Monday April 4:

            LABORATORY:      

                        Harvest, count and seed cells in chamberslides for transfection as listed in the protocol

                        Observe C2C12 culture – DAY 5.  Replace differentiation medium.

                        Start one T75 flask of cells for Thursday according to the chart below:

 

Cell Line

Seeding Density

Group I

C3H10T1/2

2.2 x 105

Group II

HeLa

7.5 x 105

Group III

NIH3T3

6.5 x 105

Group IV

C2C12

2.2 x 105

 

 

 

 

 

 

 

Tuesday April 5:

            LECTURE:

                        MyoD & Indirect immunofluorescence

                        Scientific Paper Discussion – Davis, et al. 1987 “Expression of a Single Transfected cDNA Converts Fibroblasts to Myoblasts”

 

            LABORATORY:

                        Transfect cells with myoD or pEGFP-N2 using LipofectAMINE & PLUS

                        Observe C2C12 culture – DAY 6.  Replace differentiation medium.

 

Wednesday April 6:

            LABORATORY:      

                        Change differentiation medium on chamberslides

                        Observe C2C12 culture – DAY 7.  Replace differentiation medium.

                       

Thursday April 7:

            LECTURE:

                        QUIZ (in lab)

 

            LABORATORY:        

                        Subculture your cell line (make only one T75)

                        Fix cells and complete the indirect immunofluorescence protocol, store slides at 4°C

                        Observe C2C12 culture – DAY 8 – final day

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WEEK 13

Tuesday April 12:

            LECTURE:

                        Scientific Papers Discussion

                        Lattanzi, et al. 1998 “High Efficiency Myogenic Conversion of Human Fibroblasts by Adenoviral Vector-mediated MyoD Gene Transfer”   

                        Theise and Krause 2002 “Toward a new paradigm of cell plasticity”    

 

            LABORATORY:        

                        Subculture your cell line (make only one T75)

                        View transfection immunofluorescence slides.

 

Thursday April 14:

            LECTURE:

                        Cell Fate Switching

 

            LABORATORY:

                        Subculture your cell line (make only one T75)

                        Obtain 24-well plate with C2C12 cells to differentiate, make cell morphology observations, switch medium according to protocol – this is DAY 1     

                        View transfection immunofluorescence slides.

 

Friday April 15:

            LABORATORY:

                        Make cell morphology observations on C2C12 cells in the 24-well plate and change medium – DAY 2.

 

Saturday April 16:

            LABORATORY:

                        Make cell morphology observations on C2C12 cells in the 24-well plate and change medium – DAY 3.

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WEEK 14

Sunday April 17:

            LABORATORY:

                        Make cell morphology observations on C2C12 cells in the 24-well plate and change medium – DAY 4.

 

Monday April 18:

            LABORATORY:

                        Make cell morphology observations on C2C12 cells in the 24-well plate and change medium – DAY 5.

 

Tuesday April 19:

            LECTURE:

                        QUIZ

                        Karyology

                        Scientific Paper Discussion – Katagiri, et al. 1994 “Bone Morphogenetic Protein-2 Converts the Differentiation Pathway of C2C12 Myoblasts into the Osteoblast Lineage”

 

            LABORATORY:        

                        ALP staining of C2C12 cells in 24-well plate

                        Subculture your cell line for Karyology (see protocol for seeding numbers)

                        All other flasks (except karyology) can be discarded

 

Wednesday April 20:

            LABORATORY:

                        Change medium on karyology flask

 

Thursday April 21:

            LECTURE:

                        No lecture – begin lab immediately

 

            LABORATORY:

                        Add colcemid to flasks between 10 and 11AM

                        Make metaphase spreads and store slides at -20°C

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WEEK 15

Tuesday April 26:

            LECTURE:

                        Scientific Paper Discussion

 

            LABORATORY:        

                        Stain karyology slides with Giemsa, view and count chromosomal spreads

 

Thursday April 28:

            LECTURE:

                        QUIZ (in lab)

 

            LABORATORY:

                        View and count chromosomal spreads

                        Prepare for formal presentation

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WEEK 16

Tuesday May 3:

            LECTURE: 

                        FORMAL PRESENTATIONS – Immunofluorescence, Cell Fate Switching & Karyology – in Stafford 301

 

Monday May 9:

            FINAL EXAM – 8AM