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Immunochemistry kit常見問題集
Q1:
FLICA, Tunel assay and Annexin V有什麼不同呢?
|
FLICA |
Tunel assay |
Annexin V |
|
Caspase
屬於半胱氨酸蛋白酶，當細胞接受來自藥物或細胞激素（TNF,
IFN）
的刺激，將引發細胞內死亡訊息的傳遞。FLICA為螢光探針，由caspase的inhibitor
sequence連接上綠色(FAM)或是紅色(SR)螢光,只有活化的caspase會以共價鍵和FLICA結合而保持綠色或是紅色的螢光。簡單的說FLICA是利用偵測具活性的caspase來判定細胞是否進入apoptosis。 |
細胞凋亡時，DNA被內源性核酸內切酶降解，產生帶有3′末端的切口、或斷裂DNA片段。在末端脫氧核酸轉酶(TdT)作用下，用標有螢光染劑的特殊核苷酸，如FITC-dUTP，連接到DNA片段的3′末端，觀察螢光強度即可得知DNA片段含量。 |
在細胞進行凋亡早期時，磷脂絲氨酸（PS）會外露於細胞膜外，Annexin
V對磷脂絲氨酸(PS)有親和性，將Annexin
V標定螢光物質後，可使用螢光顯微鏡或流式細胞儀偵測細胞凋亡的發生。 |
|
Label apoptotic cell內活化的caspase |
Label斷裂的DNA片段 |
Annexin V bind to外露於細胞膜外的磷脂絲氨酸（PS） |
| |
DNA斷裂片段可能是由Apoptosis來亦可能是由oxidative
stress 來,所以只看Tunel
assay無法正確判定細胞是否進行apoptosis |
Annexin V只能指出細胞表面上有磷脂絲氨酸（PS）,然而部分細胞表面一直都有PS存在,若以Annexiv
V處理無法準確判斷細胞是否正在進行apoptosis且Annexin
V無法label apoptosis過程中重要的caspase |
|
1~4
小時 |
1天(操作程序繁瑣) |
1~4小時 |
|
流式細胞儀,螢光顯微鏡,plate
reader |
流式細胞儀 |
流式細胞儀,螢光顯微鏡, |
Q2: ICT的Probe是否會影響細胞生長?
A: ICT的probe是無毒性的,所以不會影響細胞生長
Q3:
使用FLICA及Magic
Red需要先lyse
cell嗎?
A:
不需lyse
cell,
ICT的probe全是cell-permeant的,只要加到media即能擴散進入細胞與活化的caspase反應!!!
Q4:
FAM及SR的螢光可維持多久呢?
A:只要在螢光未暴露到光線下,螢光可永久維持
Q5:
ICT kit是否可使用在pre-embedded
or pre-fixed tissues or cells上?
A: ICT的kit無法使用在
pre-embedded or pre-fixed tissues or cells,
ICT
kit只能使用在fresh
or frozen samples上!!!
Q6:ICT kit包含什麼呢?
FAM-FLICA kits contain:
the caspase-specific reagent (such as FAM-VAD-FMK)
formaldehyde-based fixative
wash buffer at 10X
Hoechst stain (a blue nuclear stain)
Propidium Iodide (a red DNA stain)
SR-FLICA
kits contain: the
caspase-specific reagent (such as SR-VAD-FMK)
formaldehyde-based fixative
wash buffer at 10X
Hoechst stain (a blue nuclear stain).
Magic
Red kits contain:
the caspase- or cathepsin-specific reagent (such as MR-RR2)
Hoechst stain (a blue nuclear stain)
Acridine Orange (an orange/green lysosomal stain).
Q7:
ICT kit有25及100個test的包裝,那1個test是怎麼定義的呢?
A:
1個test定義為:使用35
mm dish,每個dish置入300ul
cell ,細胞密度
<
1x106 cells /mL
Q8:使用ICT
kit需要波長是多少呢?
|
Produst |
Excite (nm) |
Emit (nm) |
|
FAM-FLIVO |
492 |
520 |
|
SR-FLIVO |
565 |
600 |
|
FAM-FLICA |
490 |
530 |
|
SR-FLICA |
560 |
590 |
|
Magic Red |
540-590 |
>610 |
Q9:
suspension cells和
adherent cells都可以用ICT
kit嗎?
A:
ICT kit可使用在suspension
cells和
adherent cells上
Q10:請問有high
throughput screening 的apoptosis
kit嗎?
A:
有,
Magic Red Caspase 3&7 kit
非常適合screening.
只要先Transfer
cells到96
well黑盤上,
加入Magic
Red試劑培養至少15分鐘,即可開始觀察螢光變化
。
Q11: ICT
kit為何選擇FAM及SR作為label的螢光?
A:
因著FAM較FITC更亮,所以選擇FAM
且FAM及SR沒有licensing的問題
Q12: FLIVO
是否可和
Animal image (Xenogen ivis system)一起使用?
A:
FAM-FLIVO and SR-FLIVO can be imaged non-invasively with certain whole animal
imaging systems, such as the
Xenogen
IVIS system
with the
newest software package and the CRI Maestro.
Q13: ICT
是否有單賣染劑?
A:有,以下是各種染劑的規格
|
Catalog# |
Product |
Size |
|
638 |
Propidium Iodide |
1
mL(250 ug/ml) |
|
639 |
Hoechst 33342 |
1
mL(200 ug/ml) |
|
6130 |
Acridine Orange |
0.5 mL |
|
6162 |
CFSE |
250 tests |
|
6163 |
7-AAD |
125 tests |
|
6244 |
DAPI |
10
mg |
Q14: ICT是否有賣caspase的抗體呢???
A:
有,以下是各種抗體的規格
|
Catalog no. |
Product |
Size |
|
6177 |
Anti-caspase-1 |
100 ul |
|
6178 |
Anti-caspase-2 |
100 ul |
|
6179 |
Anti-caspase-3 |
100 ul |
|
6180 |
Anti-caspase-4 |
100 ul |
|
6181 |
Anti-caspase-5 |
100 ul |
|
6182 |
Anti-caspase-7 |
100 ul |
|
6183 |
Anti-caspase-8 |
100 ul |
|
6184 |
Anti-caspase-9 |
100 ul |
|
6185 |
Anti-caspase-11 |
100 ul |
|
6186 |
Anti-caspase-14 |
100 ul |
|
6187 |
Anti-cathepsin-B |
100 ul |
|
6188 |
Anti-cathepsin-D |
100 ul |
|
6189 |
Anti-cathepsin-G |
100 ul |
|
6190 |
Anti-cathepsin-H |
100 ul |
|
6191 |
Anti-cathepsin-L |
100 ul |
Q15:
有三種kit可偵測caspse
3&7, 請問這三種kit有何差異呢?
A:
|
Caspases 3&7 Kits |
FAM-FLICA™ |
SR-FLICA™ |
Magic Red™ |
|
25-tests, cat no. |
catalog # 93, $164 |
catalog # 931, $179 |
catalog # 935, $154 |
|
100-tests, cat no. |
catalog # 94, $459 |
catalog # 932, $489 |
catalog # 936, $389 |
|
fluorescence color |
green, carboxyfluorescein (FAM) |
red, sulforhodamine (SR) |
red, cresyl violet (MR) |
|
reagent structure |
inhibitor-based, FAM-DEVD-FMK |
inhibitor-based, SR-DEVD-FMK |
substrate-based, MR-(DEVD)2 |
|
assay |
add reagent to media, incubate 1-4 hours, wash cells,
analyze, end-point assay |
add reagent to media, incubate 1-4 hours, wash cells,
analyze, end-point assay |
add reagent to media and analyze,
watch fluorescence develop over time (1-72 hours) |
|
wash or no wash |
wash |
wash |
no wash |
|
wavelengths |
excitation 490, emission 520 |
excitation 560, emission 600 |
excitation 540-590, emission >610 |
|
instruments |
microscope, plate reader, FACS |
microscope, plate reader, FACS |
microscope or plate reader, special FACS |
|
backgrounds |
detects background apoptosis, typically occurring in 2-8% of
all cells |
detects background apoptosis, typically occurring in 2-8% of
all cells |
detects background apoptosis, typically occurring in 2-8% of
all cells |
|
sensitivity |
positives typically 3-10x negative controls |
positives typically 3-15x negative controls |
positives typically 2-5x negative controls |
|
best for |
microscope pictures, quantitative data with FC or plate
reader, dual staining with PI or 7AAD for necrotic cells,
Hoechst, DAPI |
microscope pictures, quantitative data with FC or plate
reader, dual staining with 7AAD for necrotic cells, Hoechst,
DAPI, GFP |
microscope pictures to watch fluorescence develop;
quantitative data with plate reader, dual staining with
green reagents |
|
cells tried |
Jurkat
HL-60
THP-1
B-Lymphocytes
Macrophages
Adherent Retinal epithelial
Adherent lung carcinoma
Squamous A431 cells head and neck carcinoma
Adherent Human pulmonary MRC-5 fibroblasts
Human coronary endothelial cells
Prostate primary culture endothelial cells PrEC
Adherent Keratoconus corneal fibroblasts |
Jurkat
HL-60
THP-1
Adherent Human pulmonary MRC-5 fibroblasts |
Jurkat
HL-60
THP-1
Fibroblasts
UMUC3 metastatic bladder cancer
MCF-7 breast cancer
U937
Adherent endothelial cells |
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