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Cell Biology Lab Cell subculture

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Cell Biology Lab Cell subculture PCB 4023 L TA: Sushmita Mustafi * * * * * * * * * * * Objectives: What is cell culture Importance How to get cells for ex vivo ... – PowerPoint PPT presentation

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Title: Cell Biology Lab Cell subculture


1
Cell Biology Lab Cell subculture
  • PCB 4023 L
  • TA Sushmita Mustafi

2
Objectives
  • What is cell culture
  • Importance
  • How to get cells for ex vivo culture
  • Primary and secondary culture
  • Physical/chemical requirements for cell culture
  • Subculture splitting
  • Established cell lines
  • J774-Eclone cells and their specifications
  • 3T3 cells
  • How to work in Biosafety cabinet.

3
What is cell culture?
  • Prokaryotic/ Eukaryotic cells are grown under
    controlled condition.
  • Why?
  • Cellular and molecular studies.
  • Protein over expression/ structure and function
    studies
  • Drug development
  • Vaccine test.

4
How to isolate cells to grow them under
controlled condition?
  • Apart from blood . cells are attached in tissue
    with the help of extracellular matrix.
  • Different enzymes like Trypsin/ pronase/
    collagenase are used to break down the matrix
    and isolate the cells
  • Explant culture Harvesting tissue to collect
    cells.

5
Cell line can be two types
  • PRIMARY CELL LINE
  • Directly from subject
  • Finite, short life span.
  • Very specific in individual research labs
  • SECONDARY CELL LINE
  • From tumor / cancer cell line
  • Infinite/ can be stored and cultured for years.
  • Major established cell lines, commercially
    available.

6
How to maintain cell lines
7
Physical condition..Incubator
  • -Provides the ideal environment for cells.
  • -Work to control 3 essential variables.
  • (1)Temperature 37C
  • (2)CO2 content 5 CO2
  • (3)Humidity 95
  • (4) pH 7.2 to 7.5

8
Chemical conditionsMedia
  • Liquid designed to support the growth of your
    cells.
  • There are different types of media for different
    types of cells.
  • AMEM
  • DMEM
  • RPMI

9
What's in that media?
  • Growth medium
  • 1) Bulk ions Sodium/ potassium/ calcium/
    Magnesium etc.
  • 2)Trace element Iron/ Zn/ Se.
  • 3) Sugar.
  • 4) Amino acids.
  • 5) Vitamins.
  • 6) Choline/ inositol.
  • 7) Serum with growth hormones.
  • 8) Antibiotics.

10
Working with cell lines
11
What do we need?
  • Cell Line
  • Flask
  • Media
  • incubator
  • Microscope
  • Biological Safety Cabinet (Hood)

12
Flasks and Plates
13
Confluency?
  • Approximately the amount of space inside the
    tissue culture flask being covered by cells.
  • Expressed as , like 50 confluency.
  • Cell growth and increase in number increases
    confluency
  • High confluency could be harmful for cells.

14
Why?
  • Accumulation of apoptotic/ necrotic cells
  • Cell to cell contact stimulate cell cycle arrest

15
What to do?Split cells.
  • Add Trypsin / using pipette of hitting the flask
  • detach cells from the adherent surface of flask.
  • Use a new flask
  • Add fresh media to the new flask
  • Introduce cells to the new flask with fresh media
    in 51 ratio.
  • Each time you split, you are making a new
    passage u should keep a note of passage number.
    Label them as P1/P2 etc..
  • Why is it important????

16
Some important cell line
  • Human
  • Hela - Cervical cancer
  • MCF-7 Breast cancer
  • Mouse
  • 3T3-L1 fibroblast
  • RAW
  • J774-Eclone

17
Our cell line
  • J774-Eclone Mouse macrophage cell line.
  • Popular in phagocytosis studies and other
    immunological studies.
  • Media Used DMEM with 10 FBS, 1 non essential
    amino acid, 1 sodium Pyruvate,
  • 1 pen/strep.

18
3T3L1
  • 3T3-L1
  • Mouse embryonic fibroblast- adipose like cell
    line.
  • Derived from swiss 3T3 mouse.
  • Used as pre adiposites
  • Media DMEM with FBS.

19
Biological Safety Cabinets
  • Leak-tight construction, HEPA filters, and
    airflow patterns work to protect product,
    personnel, or both.

20
Inverted Microscope
  • - Light source and condenser are on the top of
    the stage, while the objectives and turret are
    below.
  • -Allows for better observation of living cells at
    the bottom of a large container

21
Contamination
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