Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence of tissue homeostasis. The apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of plasma membrane is one of the earliest features. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes including FITC. This format retains its high affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells that are undergoing apoptosis. Since externalization of PS occurs in the earlier stages of apoptosis, FITC Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation.
Components
Part ? No.
Description
Size
51-65874X
FITC ? Annexin V
0.5 ? mL (1 ea)
51-66211E
Propidium ? Iodide Staining Solution
2 ? mL (1 ea)
51-66121E
10X ? Annexin V Binding Buffer
50 ? mL (1 ea)
PREPARATION AND STORAGE
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
PRODUCT NOTICES
1.Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2.Source of all serum proteins is from USDA inspected abattoirs located in the United States.
3.Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
4.Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
BD Pharm Lyse™ lysing solution is a buffered, concentrated (10X) ammonium chloride-based lysing reagent. When diluted to a 1X concentration and used as recommended, BD Pharm Lyse™ lyses red blood cells following monoclonal antibody staining. The lysing solution results in good light scatter separation of lymphocytes and red blood cell debris when analyzed by flow cytometry.??BD Pharm Lyse™ does not contain a fixative agent, so leukocytes remain viable after red blood cell lysis.
1X diluted solutions may be stored at 4° C for up to 30 days.
推薦的實(shí)驗(yàn)流程
Preparation of 1X lysing solution:??Dilute the 10X concentrate 1:10 with distilled water. The pH of the 1X solution should fall within the range of pH 7.1-7.4.??Adjust the pH if necessary. Warm the 1X solution to room temperature prior to use. 100 ml of 10X concentrate will yield a quantity of 1X solution that is sufficient to lyse 500 samples.
Lysing procedure:
Note: The following procedure is only applied to human whole blood red blood cell lysis. Since applications vary, for other samples such as mouse spleen red blood cell lysis (lysing incubation time up to 3 minutes at 37°C), bone marrow red blood cell lysis, each investigator should optimize the condition to obtain optimal results.
1. Add 2.0 ml??of 1X lysing solution to each tube containing up to 200 μl of a whole blood plus monoclonal antibody mixture.
2. Gently vortex each tube immediately after adding the lysing solution.
3. Incubate at room temperature, protected from light, for 15 minutes.
4. Centrifuge 200 X g for 5 minutes.
5. Carefully aspirate supernatent, without disturbing pellet.
6. Add 2.0 ml 1X PBS containing 1% heat-inactivated fetal bovine serum and 0.1% sodium azide (PBS-FBS).
7. Centrifuge at 200 X g for 5 minutes.
8. Carefully aspirate supernatent, without disturbing pellet.
9. Resuspend pellet in 0.5 ml PBS-FBS or a fixative such as 2% formaldehyde for flow cytometric analysis.
Troubleshooting:?Incomplete lysis may occur for several reasons: (1) The age of the specimen may affect red cell lysis, (2) The 1X lysing solution may not have been warmed to room temperature, or (3) After adding the lysing solution, the samples may not have been vortexed sufficiently. If incomplete lysis occurs (recognized by the presence of an excessive amount of visible red blood cells in the final, washed cell suspension), repeat lysing procedure. However, lysing more than two times is not recommended.
BD FACS™ lysing solution is intended for lysing red blood cells following direct immunofluorescence staining of human peripheral blood cells with monoclonal antibodies prior to flow cytometric analysis.
BD FACS lysing solution is appropriate for use with reagents such as BD Tritest™ or BD Simultest™ reagents and a suitably equipped flow cytometer. It may be used in both lyse/wash and lyse/no-wash procedures.
BD FACS™
Human
Buffered solution with <15% formaldehyde and <50% diethylene glycol.
BD FACS™ lysing solution is intended for lysing red blood cells following direct immunofluorescence staining of human peripheral blood cells with monoclonal antibodies prior to flow cytometric analysis.
BD FACS lysing solution is appropriate for use with reagents such as BD Tritest™ or BD Simultest™ reagents ａnd a suitably equipped flow cytometer. It may be used in both lyse/wash ａnd lyse/no-wash procedures
PREPARATION AND STORAGE
BD FACS lysing solution (10X) is stable until the expiration date shown on the bottle label when stored as directed. Do not use this reagent if discoloration occurs or a precipitate forms.
Components