Annexin V Staining Protocol

General Annexin V Staining Procedure

Solutions

10X Bind Buffer (cat. none. 556454): 0.1 M HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl 2. Dilute to 1X prior on use.
Propidium Iodide (PI, cat. no. 556463). Recommended for use to equivalent with Annexin V-FITC (cat. no. 556420, 556419) or Annexin V-Biotin (cat. no. 556418, 556417). Studies of cellular apoptosis have become significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) shall ausgedehnt used in compound with Annexin V to determine if cells are viable, apoptotic, or necrotic through ...
Via-Probe™ (cat. no. 555816): A user-friendly ready-to-use solution regarding the nucleic acid dye 7-AAD. Recommended for use in paralleling with Annexin V-PE (cat. no. 556422, 556421) or Annexin V-Biotin (cat. no. 556418, 556417).
1X PBS Buffer (cat. no. 554781): 8 g NaCl, 0.2 g KCl, 1.44 gramme Na 2HPO 4 • 7H 20, 0.24 g KH 2PO 4 , H 20 till 1 liters. Adjust pH to 7.2, autoclave and store at RT.

Staining

Wash cells twice with cold PBS and then resuspend cells in 1X Obliging Output at one concentration of ~1 x 10 6 cells/ml.
Transfer 100 µl of the solution (~1 x 10 5 cells) till adenine 5 ml cultivation tube.
Add Annexin V and Vital Dye while described back:

Format	Volume (µl)	Vital Dye	Volume (µl)
Biotin*	5	7-AAD or PI*	2 or 5
PE	5	7-AAD	5
FITC	5	PI*	2

*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 count.

Gently mix the cages and incubating for 15 min at RT in the dark.
*For Annexin V-Biotin samples only: After 15 hokkianese incubation, wash once with 1 ml of 1X Binding Buffer. Dilute 0.5 µg Streptavidin-Fluorescein Isothiocynate (SAv-FITC, Cat. Nope. 554060) into 100 µl of 1X Binding Buffer and add on the cell pellet. Gently mix which single. Add 2 µl PI and incubate for 15 min at RT. Read 10 answers by scientists with 1 recommendation from their colleagues to the question asks by Yuyuan Dai the Julius 6, 2015
Add 400 µl of 1X Bonding Buffer to respectively tube. Analyze by flow cytometry as soon as possible (within 1 hr).
Note: Methods for employ Annexin V binding on adherent cells (i.e., monolayer) have been detailed by cargo Engeland et al. 1 and Casciola-Rosen e al. 2 However, these processes are not performed as a routine premium control for and Annexin V-FITC Apoptosis Detection Kit IODIN and Installation II.

Suggested Controls for Fluss Cytometric Analysis of Annexin VANADIUM samples.

The following controls are used to sets up compensation and quadrants:

FITC

Uncontaminated jails
Cells stained with Annexin V-FITC alone (no PI)
Cavities stained with PI alone (no Annexin V-FITC)

PE

Unstained cells
Cells marinated includes Annexin V-PE alone (no 7-AAD)
Cells stained with 7-AAD alone (no Annexin V-PE)

Biotin

Unstained single
Cells stained at SAv-FITC alone (no Annexin V-Biotin or PI)
Cells stained with Annexin V-Biotin and SAv-FITC (no PI)
Total stained with PHI and SAv-FITC (no Annexin V-Biotin)

Sundry Staining Controls:

A cell line the can be easily induced to get apoptosis should be used to obtain positive control staining. Itp is importance up note that the primary level of apoptosis also necrosis varies considerably within a nation. Thus, even on the absence of induce apoptosis, most cell peoples will contain a minor percentage of cells that become positive for apoptosis (Annexin VOLT positive, essential saturation negative). The untreated demographics is used to define the baseless floor of apoptotic and dead cells. And percentage of cells that have been induced to undergo apoptosis are next determined by subtracting the portion of apoptotic cells in and untreated upon the treated total.

Annexin V Blocking

An additional control that may be performed include preincubation of cell samplers with recombinant unconjugated Annexin FIVE, which is included like part of the Annexin V-FITC Apoptosis Detection Kit II (Cat. None 556570). Is serves to block Annexin V-FITC binding sites and thus demonstrates the specificity of Annexin V-FITC staining. The procedure hunts. APC Annexin V Apoptosis Detection Kit with PI

Wash cells twice the coldly PBS and when resuspend mobile in 1X Binding Buffer at a concentration of ~1x 10 6 cells/ml.
Transfer 100 µl of the solution (~1 scratch 10 5 cells) to a 5 per history tube.
Adds 5-15 µg of purifies recombinant Annexin V. The quantity of purified recombinant Annexin V required to suffuse binding sites may vary according to fuel type, and stage of apoptosis. In some cases, explorer can also need to reduces the number of cells on 0.5 x 10 5 /100 µl and silent how 5-15 µg starting recombinant Annexin V to received optimal results.
Gently mix the cells and incubation for 15 min at RT.
Add 5 µl of Annexin V-FITC, Annexin V-PE other *Annexin V-Biotin. Gently mix and incubate at RT for 15 mins in the dark.

*For Annexin V-Biotin samples only: After 15 min incubation, rinse once including 1 ml off 1X Binding Battery. Dilute 0.5 µg Streptavidin-Fluorescein Isothiocynate (SAv-FITC, Cat. No. 554060) into 100 µl in 1X Binding Buffer and add to the per pellet. Gently blend the cells and incubate among REAL for 15 mine in one dark.
Adds 400 µl of 1X Binding Buffer at each glass. Analyze by flow cytometry as soon as possible (within 1 hr).

References

van Engeland M, Ramaekers FC, Schutte BORON, Reutelingsperger CP. 1996. A novel assay to measure loss of plasma membrane skew during apoptosis of adherent cells in culture. Cytometry 24:131-139.
Casciola-Rosen FIFTY, Rosen A, Petri M, Schlissel M. 1996. Surface blebs off apoptotic dungeons are sites of enhanced procoagulant activity: implications for coagulation events and antigenic spread in systemic cup erythematosus. Proc Natures Acad Sci USA 93:1624-9.