Theory of the practical
Practical – research aim
- What is the effect of ALCAM-ALCAM interactions on MMP2 activation
- What is the effect of ALCAM-ALCAM interactions on gene expression (ALCAM responsive genes)
Cell culture
- BLM-Cells
o Expressing normal ALCAM
o Over-expressing truncated ALCAM (D1 and D2 domain deleted, dN ALCAM)
o Over-expressing soluble ALCAM (only expressing D1, sALCAM)
- 100% confluent T75 = 7.5 * 10^6 cells
o (= 100.000 cells / cm2 )
- Investigate cell-cell / ALCAM-ALCAM interactions
o Culture @ different densities
o 3D culture → 104 , 105 , 106 cells per collagen gel
o Gene expression analysis → 60% confluent (low) & 200% confluent (high)
Passaging cells
- ~80% density
- Passage 1/5
, Cell culture – passaging adhering cells
- BLM cells: Adherend (dN and S different medium!)
- Remove medium, wash with PBS (do not ‘pipet on top of them’)
- Trypsin (keep at RT), add 1ml, incubate 2-3 min @ 37C
- Add medium with FCS
- Resuspend vigorously
- Transfer to tube
- Count the cells
- Does the number of cells correspond to what you estimated initially?
- Passage at 1/2, 1/5, … (depends on confluency!)
Example T75
- 80 % density, passage 1/5th to a new flask
- All cells were resuspended in 6.5 ml (1,5ml trypsin + 5 ml D10)
- Take 1.3 ml of cell suspension • Place back in the old flask:
- Top off with medium (add ~ 9 ml)
- Devide cell suspension equally over the bottom
CAPS of flasks
- With filter
- Without filter
- Gas exchange! Open cap a bit!
Cell culture – 3D culture
- MMP2 activation requires cell-matrix interactions
o Pro-MMP2 and active MMP2
- type I Collagen present
- Wells plate with collagen and on top you grow cells
o Cells grow on collagen
- Investigate MMp2 activation
- MMP2 is expressed in probe form pro-MMP2
o For activation pro domain needs to be cut of
- Activation requires ECM interaction with collagen
MMP2
- Matix metallo protease MMP2 is a protease
- Cleves extra cellular matrix CLEVEAGE collagen
- Calcium dependent
- Substrate gelatin (derived form collagen)
Practical – research aim
- What is the effect of ALCAM-ALCAM interactions on MMP2 activation
- What is the effect of ALCAM-ALCAM interactions on gene expression (ALCAM responsive genes)
Cell culture
- BLM-Cells
o Expressing normal ALCAM
o Over-expressing truncated ALCAM (D1 and D2 domain deleted, dN ALCAM)
o Over-expressing soluble ALCAM (only expressing D1, sALCAM)
- 100% confluent T75 = 7.5 * 10^6 cells
o (= 100.000 cells / cm2 )
- Investigate cell-cell / ALCAM-ALCAM interactions
o Culture @ different densities
o 3D culture → 104 , 105 , 106 cells per collagen gel
o Gene expression analysis → 60% confluent (low) & 200% confluent (high)
Passaging cells
- ~80% density
- Passage 1/5
, Cell culture – passaging adhering cells
- BLM cells: Adherend (dN and S different medium!)
- Remove medium, wash with PBS (do not ‘pipet on top of them’)
- Trypsin (keep at RT), add 1ml, incubate 2-3 min @ 37C
- Add medium with FCS
- Resuspend vigorously
- Transfer to tube
- Count the cells
- Does the number of cells correspond to what you estimated initially?
- Passage at 1/2, 1/5, … (depends on confluency!)
Example T75
- 80 % density, passage 1/5th to a new flask
- All cells were resuspended in 6.5 ml (1,5ml trypsin + 5 ml D10)
- Take 1.3 ml of cell suspension • Place back in the old flask:
- Top off with medium (add ~ 9 ml)
- Devide cell suspension equally over the bottom
CAPS of flasks
- With filter
- Without filter
- Gas exchange! Open cap a bit!
Cell culture – 3D culture
- MMP2 activation requires cell-matrix interactions
o Pro-MMP2 and active MMP2
- type I Collagen present
- Wells plate with collagen and on top you grow cells
o Cells grow on collagen
- Investigate MMp2 activation
- MMP2 is expressed in probe form pro-MMP2
o For activation pro domain needs to be cut of
- Activation requires ECM interaction with collagen
MMP2
- Matix metallo protease MMP2 is a protease
- Cleves extra cellular matrix CLEVEAGE collagen
- Calcium dependent
- Substrate gelatin (derived form collagen)
