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Viabilitas sel, proliferasi sel dan banyak fungsi sel

penting transduksi-bisa menjadi dirangsang atau


dipantau dengan berbagai bahan kimia dan
biologis reagen
Antara lain : apoptosis, adhesi sel, chemotaxis,
endositosis, sekresi dan sinyal sel

Mekanisme sel seperti radikal intraseluler,


Konsentrasi free-ion, atau potensial membran yang
telah dapat teramati dengan tepat menggunakan
pewarnaan responsif fluorescent indikator.
VIABILITAS :
Sel-sel Yang Mampu Berkembang
Dalam Lingkungan Kultur

Pengukuran Viabilitas Sel (Cells Viability Measurement) :


Kelangsungan hidup adalah ukuran
Keadaan metabolik populasi sel yang merupakan
indikasi potensi pertumbuhan
Metode yang umum digunakan dalam viability
meansurement :

o Integritas Membran
• Trypan blue exclusion: membran yang bocor
memungkinkan reagent masuk ke dalam sel
• Pengamatan dapat dengan mikroskop
manual
• Keberhasilan tergantung ketrampilan
o Cellometer Solution:
• Menggunakan alogaritma perangkat lunak

• Mengurangi ketergantungan operator

• Mngurangi kesalahan perhitungan

• Mempercepat penghitungan

• Pengelolaan data yang lebih baik


Trypan Blue Viability for Cell Lines: Exclusion Assay

H9 LCL

M19 Raw

K562 T cell

Cellometer has been used for more than 300 cell lines
Count Live Cells and Measure Viability with Fluorescein
Diacetate (FDA)
Assay principle
Fluorescein diacetate (FDA) Esterases Fluorescein
(non- fluorescence) (Fluorescence)

Cellometer images and analysis

Cellometer cell sample analysis reports


•Total # of cells: Cell concentration measurement
•Count FL positive cells: live cell concentration measurement
•Calculate cell viability automatically
•Cell size measurement and population size histogram display
•Save all cell images
Nexcelom.com Online Resources
info@nexcelom.com | 978.327.5340 Cellometer Simply
® Counted
Count and Determine Viability of Yeast
Using Oxonol, Cellometer Vision 10x

Green outline: all yeast cells Green outline: dead cells, oxonol permeable
Red outline: live yeast cells

Automatically generate live cell concentration Automatically generate live cell size histogram
and viability
Nexcelom.com Online Resources
info@nexcelom.com | 978.327.5340 Cellometer® Simply
Counted
Fluorescence Based Cell Viability Methods

o Membrane Intregity
• Pewarna binding-DNA (tergantungan
permeabilitas)
• AO (acridine orange): permeabel untuk sel hidup
dan mati
• PI (propidium iodida), EB (Ethidium bromide), 7-
AAD: (tidak permeabel untuk sel hidup )
• Lainnya: SYTO ® 13, SYTO 24 ®, SYTO® 14
Live Cell Concentration and PI Viability: Cell Lines

Data output:
• Total # of cells, FL+ cells
• Cell concentration
• Live cell concentration
• Viability
• Cell images
• Cell size histogram

Sample Cell Concentration Viability %


Jurkat 1.7 x 106/ml 91%
Mouse thymocyte 1.6 x 106/ml 81%
Mouse splenocyte 1.1 x 106/ml 85%
Mouse bone marrow 0.95 x 106/ml 81%

Other dyes: EB, 7-ADD


Nexcelom.com Online Resources
info@nexcelom.com | 978.327.5340 Cellometer Simply
® Counted
o Enzymatic Activity :

• Non-fluoresen esterase berdifusi ke dalam


sel

• Sel hidup dengan aktivitas enzimatik yang


kuat menghasilkan fluorescent sel hidup

• Mempertahankan produk fluorscent

• Contoh:
- Calcein-AM to calcein
- Fluorescein diacetate (FDA)to fluorescein
o Deteksi simultan dari populasi sel hidup dan
mati untuk sel line

• Kombinasi di atas dengan deteksi duo-


fluoresensi untuk setiap sel sample
Live, Dead Cell Concentrations and Viability of Primary
Cells Using Dual Staining Method
Spleenocyte: AOPI

PBMC: AOEB

Fluorescent staining helps identify


cells of interest for primary cell
samples

Nexcelom.com Online Resources


info@nexcelom.com | 978.327.5340 Cellometer® Simply
Counted
How Does It Work…

~20µL sample Add 20µL sample+20µL


fluorescent dye
Pipette 20µL of sample
Insert chamber in Brightfield and fluorescent c
into disposable
counting chamber
ell Cellometer images acq uired and analyzed

Output data
generated instantly

Nexcelom.com Online Resources


info@nexcelom.com | 978.327.5340 Cellometer Simply
® Counted
FLOW CYTOMETRY :
POLA DISTRIBUSI FASE PROLIFERASI (G0/G1, S, G2/M, Ap) PADA SEL
KUMULUS

R2
R2-5 = Untuk Penentuan Gate Fase Proliferasi

M1 R6 = Gate fase G2/M


R3
R7 = Gate fase S
R4 M2
R5 R8 = Gate fase G0/G1
M3
R9 = Gate fase Ap
M4

M1-4 = Untuk Penentuan Puncak Fase Proliferasi


Fi le: Da ta.0 01 Sam ple ID: 0 ng

Regi on Events % Ga ted % Tota l


R1 13 99 10 0.00 26 .20
R2 7 0.50 0.13
R3 56 4.00 1.05
R4 81 0 57 .90 15 .17
R5 41 8 29 .88 7.83
R6 2 0.14 0.04
R7 65 4.65 1.22
R8 84 5 60 .40 15 .82
R9 33 5 23 .95 6.27

Fi le: Da ta.0 03 Sam ple ID: 50 ng

Regi on Events % Ga ted % Tota l


R1 11 81 10 0.00 20 .88
R2 46 1 39 .03 8.15
R3 13 2 11 .18 2.33
R4 33 3 28 .20 5.89
R5 21 6 18 .29 3.82
R6 39 8 33 .70 7.04
R7 18 6 15 .75 3.29
R8 31 4 26 .59 5.55
R9 15 7 13 .29 2.78
Fi le: Da ta.0 02 Sam ple ID: 10 0 ng

Regi on Events % Ga ted % Tota l


R1 31 06 10 0.00 30 .41
R2 20 53 66 .10 20 .10
R3 21 7 6.99 2.12
R4 57 2 18 .42 5.60
R5 18 6 5.99 1.82
R6 17 86 57 .50 17 .48
R7 35 2 11 .33 3.45
R8 56 9 18 .32 5.57
R9 15 3 4.93 1.50

Fi le: Da ta.0 04 Sam ple ID: 20 0 ng

Regi on Events % Ga ted % Tota l


R1 11 62 10 0.00 28 .69
R2 67 3 57 .92 16 .62
R3 97 8.35 2.40
R4 28 7 24 .70 7.09
R5 70 6.02 1.73
R6 63 2 54 .39 15 .60
R7 13 4 11 .53 3.31
R8 27 7 23 .84 6.84
R9 41 3.53 1.01