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CFSE_assay_lecture课件
Cell division tracking using CFSE;Workshop outline:
- Introduction to CFSE.
- Some of the crucial things you need to consider.
- A basic protocol around which to plan your application.
-Coffee break?
- Question time:
*specific problems you may have been experiencing.
*discussion about any applications you are considering.
*helping other investigators at your institute.
Bruce.Lyons@imvs.sa.gov.au
(08) 8222 3590 ;The original observation;Flow Cytometric uses of CFSE;O-C-CH3;Applications;Dilution of CFSE with cell division;Properties of CFSE;CFSE Fluorescence intensity is proportional
to dye concentration at the time of staining;Using differential labelling to track survival
of different cell populations ;CFSE intensity drops rapidly in the first few days in the
absence of cell division, but then stabilises;Cell division profiles are not
affected by rapid CFSE loss
in the first few days after
staining- this loss of stain due
to catabolism is proportional.;Measurement of cell properties simultaneously with CFSE ;Division tracking of cell nuclei to examine transcription factor translocation to the nucleus (Hasbold Hodgkin 2000);Maximising resolution;Using CFSE to track CML
progenitor proliferation;Live:;Calculating precursor frequency
0 1590 /1 1590
1 1140 /2 570
2 1388 /4 347
3 687 /8 86
4 41 /16 2.6
Total; 2595
Cohort Total; 1005.6
% original cells dividing is 1005.6divided by (2595 + 1005.6) = 38.7%
;Gate on R1;Ungated;CML CD34 cells stimulated with GM-CSF; GATE GEN. EV. f #Parent
R 3 0 561 1 561
R 4 1 1158 2 579
R 5 2 2311 4 578
R 6 3 1541 8 193
R 7 4 142 16 9
SUM: 5713 1920
P.I. 5713/1920 = 2.98;GM-CSF;Lyons (1997) Immunol. Cell Biol. 75; 7;Examining cell division linked differentiation;Substantial overlap of Fluorescein emission into PE
channel makes compensation of CFSE challenging!;Compensation can be very tricky when CFSE is bright !;Fl-1 gai
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