BIO 273 Week 5 Centrifugation and Red Blood Cells
-
Upload
nipun-sharma -
Category
Documents
-
view
13 -
download
7
description
Transcript of BIO 273 Week 5 Centrifugation and Red Blood Cells
Centrifugation & Osmotic Fragility – Chapter 5
Centrifugation – Spinning solutions at high speed to separate out different components
How does it work? – Centrifugal, frictional and buoyant forces act together to separate objects according to mass and density
Frictional Force – prevents particles from moving in the medium
Buoyant Force – prevents particles from moving in the medium
Centrifugal Force – Pushes out from the centre of rotation to move particles
*When the particles reach the bottom of the tube or a steady velocity, the three forces add up to zero*
Centrifugal force - frictional force - buoyant force = zero
Centrifugation & Osmotic Fragility – Chapter 5
Determining Centrifugal Force (RCF or xg)
RPM – speed of the rotor in revolutions per minute
r – distance from the centre/axis of rotation to the end of the tube (measured in cm)
t – the time of centrifugation
RCF = [(rpm x 2π)/60]2 x (r/980)
r
Centrifugation & Osmotic Fragility – Chapter 5
https://www.thermo.com/eThermo/CMA/PDFs/Various/File_661.pdf
Table provided by centrifuge manufacturer
Centrifugation & Osmotic Fragility – Chapter 5
Nomograph
http://www.corning.com/lifesciences/us_canada/en/technical_resources/doc_library/nomogram_computing_rcf.aspx
Centrifugation & Osmotic Fragility – Chapter 5
The Experiment
We are going to be testing the effect of salt concentration on red blood cells.
Why?
Diseases such as sickle cell anemia and spherocytosis cause red blood cells to have a different shape than a healthy red blood cells. The change in shape can be detected by testing the reaction of a blood sample to salt solutions at different concentrations.
Consider what would happen to each of these cells in an isotonic solution:
Which of the cells would be most sensitive to a hypotonic solution? Least sensitive?
Centrifugation & Osmotic Fragility – Chapter 5
The Experiment
Decreasing NaCl concentrations
RBC in isotonic solution.Haemoglobin is inside the cell
RBC in hypotonic solution.Haemoglobin is released form the cell
When you centrifuge each tube, what will you find in the pellet? What will you find in the supernatant?
Centrifugation & Osmotic Fragility – Chapter 5
Preparation of red blood cell solutions at various NaCl concentrations
TubeNaCl
concentration (%)
Total Volume
(mL)
Volume of 1% NaCl
(mL)
Volume of
dH2O
(mL)
Volume of blood stock
(mL)
1 0 10
2 0.1 10
3 0.3 10
4 0.5 10
5 0.7 10
6 0.9 10
blank 0.9 10
Centrifugation & Osmotic Fragility – Chapter 5
Calculations
TubeNaCl
concentration (%)
Total Volume
(mL)
Volume of 1% NaCl
(mL)
Volume of
dH2O
(mL)
Volume of blood stock
(mL)
6 0.9 10
Volume of sodium chloride:
C1V1 = C2V2
(1%)(V1) = (0.9%)(10mL) V1 = 9mL
Volume of blood stock:
dilution x concentration = volume 1/50 x 10mL = volume 0.2mL= volume
Volume of distilled water:
Volume of water = total volume - volume of NaCl - volume of bloodVolume of water = 10mL - 9mL - 0.2mLVolume of water = 0.8 mL
Centrifugation & Osmotic Fragility – Chapter 5
Experimental considerations
1) Keep in mind that you are testing the effect of NaCl concentration on cell lysis not the effect of mechanical damage. Be gentle.
2) What order should the water, blood and sodium chloride be added? Why?
3) Should the tubes all have the same total volume? Why is this important?
4) If the cells do not lyse, will they be in the supernatant or in the pellet?
5) If the cells do lyse, what colour would the supernatant be? Why?
Centrifugation & Osmotic Fragility – Chapter 5
Calculation of % lysis:
Tube NaCl concentration (%) % Transmittance Absorbance
1 0 20% 0.70
6 0.9 90% 0.05
% lysis = x 100 absorbance of tube X absorbance of tube 1
% lysis = x 100 absorbance of tube 6 absorbance of tube 1
% lysis = x 100 0.05 0.70
% lysis = 7%
Why is the absorbance of tube 1 used as the
denominator?
Hint: what is the % lysis of tube 1?