Cell biology Lecture 9

Essential nutrients for cell culture.

Amino acids, glucose, vitamins, salts (in media such as DMEM or RPMI 1640)

Physiological ph

(around 7.2–7.4)

Physioogical Temperature

37 degrees C

Humidity of cell culture

about 95%

What do growth factors do

Stimulate cell growth and survival Provided by serum (e.g., fetal bovine serum, FBS

Antibiotics in cell cultures

Penicillin and Streptomycin

Primary cells and characteristics

Cells isolated from normal tissues. They can

grow in a culture dish for several generations, but they have

limited lifespan.

Grow in monolayer

Stop dividing when in contact of one another (contact inhibition)

Simply, how do you derive primary cells

1. Take sample of cells

2. Add digestive solution to seperate them

3. select desired cells

4. add them to culture

5. freeze using n2

Cell lines and characteristics

Cultures of animal cells that can be propagated

repeatedly and, in some cases, indefinitely.

Rapid growth rate and lost contact inhibition

Exhibit tumor morphology

The HeLa cell line

Established in 1951 from a biopsy of a cervical tumor

taken from Henrietta Lacks in 1951. These cells have been maintained and

widely used in research since their isolation.

In vitro

Experiments performed with cellular structures or tissues

isolated from living organisms, which include most laboratory

studies.

In vivo

Experiments conducted in living organisms, such as drug

testing in clinical trials.

How are sections of tissue or cells selected?

Laser capture microdissection

Laser capture microdissection

Take tissue sample with cancer or other region of interest is put on a class slide. 1st laser beam cuts around region of interest. 2nd Laser beam catapults selected region into container.

How are specific types of cells separated?

Fluorescence Activated Cell Sorter

Fluorescence Activated Cell Sorter

an instrument that sorts cells by the different charges they have after being labeled with fluorescent dyes when passing through a laser.

How are specific organelles separated

Cell Fractionation - A lab technique to separate organelles of

the cell. It usually includes two steps.

Step 1. Homogenization

(A starting material of pure cells or tissue are homogenized by physical or chemical methods)

Step 2. centrifugation

Homogenized cell content is place in a centrifuge to separate organelles based on their physical properties)

Because nuclei are large, they can be spun down at

low speeds

Magnification

the ratio of an object’s image to its actual size.

Resolution

the minimal distance of two points can be distinguished. It is a measure of clarity.

Light Microscope

Magnification: 40 - 1000x, Resolution: ~200 nm

Bright-field

Standard form; light passes directly through specimen (used with stains).

Phase-contrast

Shows variations in density of the cell.

Differential interference contrast (DIC / Nomarski)

Densities of the cell are magnefied to amplify the

differences in different areas

Dark-field

Shows bright objects on a dark background; highlights small, unstained structures.

Fluorescence

Similar to light microscope in magnification and

resolution.

• Most useful to localize a specific protein or a

structure in the cell.

• Cellular structures can be labeled with of

fluorescence probes, chemicals that emit

fluorescence

Confocal microscope

Specialized form of fluorescence microscopy. Uses lasers or special optics to selectively focus on a narrow region of specimen, thus producing sharp images.

DAPI – DNA binding dye

Emits a blue color when binded to DNA. Can permeate the cell membrane because it is hydrophobic.

Phalloidin

toxin originally isolated from the death cap mushroom that happens to bind between subunits of actin filaments. Fluorescein or Rhodamine hitch a ride with it to the actin and gives green or red fluorescence, respectively.

Immunocytochemistry – antibody-based fluorescence microscopy

1. Cells are fixed

2. You introduce a primary antibody that bunds to a protein of interest.

3. You add a secondary antibody containing a fluorescent marker that binds to the primary anti body.

4. Observe under fluorescent microscope to see location of protein of interest

GFP(Green fluorescent protein )

Genetically modified cells produce proteins with GFP attached to the end, a fluorescent protein from jellyfish, allowing scientists to visualize and track proteins in live cells under a fluorescence microscope.

Electron Microscopes (EMs)

The principles are similar to those of LMs. The difference

is that EMs use electron beam and electromagnetic lens

to generate powerful resolutions.

Magnification: 1–2 million times

Resolution: 0.1 nm (2000x higher than LM)

2 types:

Transmission electron microscope (TEM)

electron beam passes through the specimen and produces detailed internal structures

Scanning electron microscope (SEM)

electron beam scans the surface of the specimen and produces 3-dimensional image.