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Protocols
Annexin V Staining Protocol
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General Annexin V Staining Procedure
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1.
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10X Binding Buffer (cat. no. 556454): 0.1 M HEPES, pH 7.4;
1.4 M NaCl; 25 mM CaCl 2 . Dilute to 1X prior to
use.
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2.
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Propidium Iodide (PI, cat. no. 556463). Recommended for
use in parallel with Annexin V-FITC (cat. no. 556420, 556419)
or Annexin V-Biotin (cat. no. 556418, 556417).
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3.
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Via-Probe™ (cat. no. 555816): A convenient ready-to-use
solution of the nucleic acid dye 7-AAD. Recommended for use
in parallel with Annexin V-PE (cat. no. 556422, 556421) or
Annexin V-Biotin (cat. no. 556418, 556417).
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4.
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1X PBS Buffer (cat. no. 554781): 8 g NaCl, 0.2 g KCl, 1.44
g Na2HPO 4 • 7H20, 0.24
g KH2PO4 , H20 to 1 liter.
Adjust pH to 7.2, autoclave and store at RT.
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1.
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Wash cells twice with cold PBS and then
resuspend cells in 1X Binding Buffer at a concentration of ~1 x 106 cells/ml.
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2.
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Transfer 100 µl of the solution (~1 x 105
cells) to a 5 ml culture tube.
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3.
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Add Annexin V and Vital Dye as described
below:
*The optimal amount of PI may range between 2–10 µl/test depending
on cell type and experimental system. 2 µl/test is the recommended
starting amount.
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4.
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Gently mix the cells and incubate for 15 min at RT in the dark.
*For Annexin V-Biotin samples only: After 15 min incubation,
wash once with 1 ml of 1X Binding Buffer. Dilute 0.5 µg Streptavidin-Fluorescein
Isothiocynate (SAv-FITC, Cat. No. 554060) into 100 µl of 1X
Binding Buffer and add to the cell pellet. Gently mix the
cells. Add 2 µl PI and incubate for 15 min at RT.
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5.
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Add 400 µl of 1X Binding Buffer to each
tube. Analyze by flow cytometry as soon as possible (within 1 hr).
Note: Methods for utilizing Annexin V binding on adherent cells
( i.e., monolayer) have been described by van Engeland et
al.1 and Casciola-Rosen et al.2 However,
these methods are not performed as a routine quality control
for the Annexin V-FITC Apoptosis Detection Kit I and Kit II.
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Suggested Controls for Flow Cytometric
Analysis of Annexin V samples.
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The following controls are used to set up
compensation and quadrants:
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FITC
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1.
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Unstained cells
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2.
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Cells stained with Annexin V-FITC alone
(no PI)
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3.
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Cells stained with PI alone (no Annexin
V-FITC)
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PE
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1.
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Unstained cells
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2.
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Cells stained with Annexin V-PE alone
(no 7-AAD)
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3.
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Cells stained with 7-AAD alone (no
Annexin V-PE)
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Biotin
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1.
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Unstained cells
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2.
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Cells stained with SAv-FITC alone (no
Annexin V-Biotin or PI)
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3.
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Cells stained with Annexin V-Biotin and
SAv-FITC (no PI)
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4.
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Cells stained with PI and SAv-FITC (no Annexin
V-Biotin)
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Other Staining Controls:
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A cell line that can be easily induced to
undergo apoptosis should be used to obtain positive control staining.
It is important to note that the basal level of apoptosis and necrosis
varies considerably within a population. Thus, even in the absence of
induced apoptosis, most cell populations will contain a minor
percentage of cells that are positive for apoptosis (Annexin V
positive, vital dye negative). The untreated population is used to
define the basal level of apoptotic and dead cells. The percentage of
cells that have been induced to undergo apoptosis is then determined by
subtracting the percentage of apoptotic cells in the untreated from the
treated population.
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Annexin V Blocking
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An additional control that may be performed includes preincubation
of cell samples with recombinant unconjugated Annexin V, which is
included as part of the Annexin V-FITC Apoptosis Detection Kit II
(Cat. No 556570). This serves to block Annexin V-FITC binding sites
and thus demonstrates the specificity of Annexin V-FITC staining.
The procedure follows.
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1.
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Wash cells twice with cold PBS and then
resuspend cells in 1X Binding Buffer at a concentration of ~1x 106 cells/ml.
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2.
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Transfer 100 µl of the solution (~1 x 105 cells) to a 5 ml culture tube.
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3.
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Add 5-15 µg of purified recombinant
Annexin V. The amount of purified recombinant Annexin V required to
saturate binding sites may vary according to cell type, and stage of
apoptosis. In some cases, investigators may also need to reduce the
number of cells to 0.5 x 105 /100 µl and still add 5-15 µg of
recombinant Annexin V to obtain optimal results.
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4.
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Gently mix the cells and incubate for 15 min at RT.
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5.
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Add 5 µl of Annexin V-FITC, Annexin V-PE
or *Annexin V-Biotin. Gently mix and incubate at RT for 15 min in the
dark.
*For Annexin V-Biotin samples only: After 15 min incubation,
wash once with 1 ml of 1X Binding Buffer. Dilute 0.5 µg Streptavidin-Fluorescein
Isothiocynate (SAv-FITC, Cat. No. 554060) into 100 µl of 1X
Binding Buffer and add to the cell pellet. Gently mix the
cells and incubate at RT for 15 min in the dark.
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6.
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Add 400 µl of 1X Binding Buffer to each
tube. Analyze by flow cytometry as soon as possible (within 1 hr).
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References
1. van Engeland M, Ramaekers FC, Schutte B, Reutelingsperger CP. 1996. A novel
assay to measure loss of plasma membrane asymmetry during apoptosis of adherent
cells in culture. Cytometry 24:131-139.
2. Casciola-Rosen L, Rosen A, Petri M, Schlissel M. 1996. Surface blebs on
apoptotic cells are sites of enhanced procoagulant activity: implications for
coagulation events and antigenic spread in systemic lupus erythematosus. Proc
Nat Acad Sci USA 93:1624-9.
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