FITC Annexin V/PI Cell Apoptosis Kit

FITC Annexin V/PI Cell Apoptosis Kit

Product Code:F6012L

Product Specifications100T

Storage conditions:

4℃Avoid light and refrigerate, do not freeze.The expiration date can be found on the outer packaging.

Spectral characteristics:

FITC-Annexin V: Ex/Em = 494/518 nm

PI: Ex/Em = 535/617 nm (with DNA)

Product Introduction

FITC-Annexin V/PIThe apoptosis assay kit provides a fast and convenient method for labeling early apoptotic cells (green) and necrotic or late apoptotic cells(Red) Detect the level of cell apoptosis. The product can be detected using flow cytometry or other fluorescence detection equipment.

Annexin V(membrane associated protein)-V）It is a type of molecular weight35-36KDTheCa2+Dependent phospholipid binding protein, capable of binding to phosphatidylcholine（PS）Selective binding.Phosphatidyl（PS）Mainly distributed on the inner side of the cell membrane, adjacent to the cytoplasm. In the early stages of cell apoptosis, different types of cells will release phospholipidsAcyl inversion onto the cell surface, exposed to the extracellular environment. At this point, use a green fluorescent probeFITCMarked withAnnexin VThat isFITC - Annexin V，Phosphatidyl with outward rotation（PS）By combining, flow cytometry or fluorescence microscopy can be used to directly detect the important feature of phosphatidyltransferase turnover, which is an important characteristic of cell apoptosisZheng. For necrotic or late stage apoptotic cells, due to the disruption of cell integrity,FITC - Annexin VIt can enter the cytoplasm and be located inside the phospholipid layerPSCombined, it also causes necrotic cells to exhibit green fluorescence.

Propidium iodide（Propidium Iodide, PI）It's a kind ofDNACombined with dyes, it can stain the fine details of necrotic cells or cells that have lost their membrane integrity in the late stage of apoptosisNucleus.PICan be done by488、532 Or546 nmLaser excitation produces red fluorescence.

Usage:

1. Experimental design:

Blank tube: negative control group cells, without addingFITC-Annexin V/PIUsed to regulate voltage.

Single staining tube: Positive control group cells, only addedFITC-Annexin V/just addPIUsed for adjusting compensation.

Detection tube: processed cells, addedFITC-Annexin V/PIAfter adjusting the voltage compensation using blank tubes and single dye tubes, obtain the required flow data.

2. Collect cells:

(1) For suspended cells:

a.After undergoing cell apoptosis stimulation,1000 rpmcentrifugation5 minDiscard the supernatant, collect the cells, and usePBSGently resuspend the cells and count them. Note:PBSSuspension cannot be omitted,PBSThe process of resuspension also plays a role in washing cells, which can ensure the subsequent recoveryFITC-Annexin VThe combination.

b.取5×104 -1×105A suspended cell,1000 rpmcentrifugation5 minDiscard the supernatant and add100 µL 1×Annexin VGently resuspend cells in combination with buffer solution.

c.join5 µL FITC-Annexin VGently mix well.

d.join5 µL PIStaining solution, gently mix well.

e.Room temperature( 20-25ºC )Incubate in the dark10 - 15 minAluminum foil can be used to avoid light. During the incubation process, cells can be resuspended2-3To improve the dyeing effect.

(2) For adherent cells:

a.Suck out the cell culture medium into a suitable centrifuge tube,PBSWash the adherent cells once and add an appropriate amount of cell digestion solution(excludeEDTA)Digestive cells. Incubate at room temperature until gently blowing can remove the cell digestion solution when the adherent cells are blown down. Excessive digestion that needs to be avoided. Note: For adherent cells, the digestion step is crucial. If the digestion time is too short, cells need to be blown hard to detach, which can easily cause damage to the cell membrane and lead to false positives of cell necrosis; If the digestion time is too long, it is also easy to cause cell membrane damage and false positives of cell necrosis, and even affect the phosphatidylcholine on the cell membraneFITC-Annexin VThe combination interferes with the detection of cell apoptosis.

b.Add the cell culture medium collected in the previous step, gently blow down the cells, and transfer them to a centrifuge tube,1000 rpmcentrifugation5 minDiscard the supernatant, collect the cells, and usePBSGently resuspend the cells and count them. Note: Adding the cell culture medium from the previous step is very important. On the one hand, it can collect cells that have already been suspended and undergone apoptosis or necrosis. On the other hand, the serum in the cell culture medium can effectively inhibit or neutralize residual cells. The residue will be digested and degraded, and subsequently addedFITC-Annexin VResulting in staining failure.

c.取5×104 -1×105A suspended cell,1000 rpmcentrifugation5 minDiscard the supernatant and add100 µL 1×Annexin VGently resuspend cells in combination with buffer solution.

d.join5 µL FITC-Annexin VGently mix well.

e.join5 µL PIStaining solution, gently mix well.

f.Room temperature(20-25ºC )Incubate in the dark10 - 15 minAluminum foil can be used to avoid light. During the incubation process, cells can be resuspended2-3To improve the dyeing effect.

3. Result analysis:

(1) Flow cytometry detection:

a. After incubation, 400 μ L of 1 × Annexin V binding buffer can be directly added to resuspend the cells, and immediately detected on the machine. FITC Annexin V is excited by 488 nm laser, and the fluorescence emission spectrum is detected at 530 nm (FITC channel), and the PI channel emission spectrum is about 617 nm.

b. On the scatter plot of the bivariate flow cytometer, live cells are shown in the lower left quadrant as FITC Annexin V -/PI -; The lower right quadrant represents early apoptotic cells, which are FITCAnnexin V+/PI -; The upper right quadrant shows necrotic and late stage apoptotic cells, represented by FITC Annexin V+/PI+; The upper left quadrant displays naked nuclear cells, which are FITC Annexin V -/PI+.

(2) Fluorescence microscopy detection:

a. Centrifuge at 1000 rpm for 5 minutes, collect cells, and gently resuspend them in 400 µ L of 1 × Annexin V binding buffer. Transfer the cells to a 96 well plate and settle for a moment or perform cell smear, then observe under a fluorescence microscope.

b. FITC Annexin V can use FITC compatible filters, while PI can use Cy3 or Texas compatible filters.

Notes:

1. Before use, please centrifuge the product instantly to the bottom of the tube before conducting subsequent experiments.

2. To reduce the process of cell apoptosis, the incubation process can be performed on ice, but the incubation time should be extended at least until30 min

3. Due to the rapid process of cell apoptosis, it is recommended that the sample be stained1 hAnalyze internally.

4. For adherent cells, digestion is a crucial step. When adherent cells induce apoptosis, if there are floating cells, they need to be collected and stained together with adherent cells. Be careful when handling adherent cells and try to avoid human damage to the cells as much as possible. The digestion time is too short, and cells need to be blown hard to detach, which can easily cause damage to the cell membrane,PIExcessive intake; If the digestion time is too long, the cell membrane is also prone to damage, and it may even affect the phosphatidylcholine and phosphatidylcholine on the cell membraneFITC-Annexin VThe combination. After covering the bottom of the well plate during digestion, gently shake it to fully contact with the cells, then discard most of it and use the remaining small amount to digest for a period of time. Wait until the intercellular gaps increase and the bottom of the bottle becomes spotted, then stop. Try not to use it in digestive juices as much as possibleEDTA，EDTAIt will affectAnnexin VWithPSThe combination.

5. After digesting the adherent cells, it is recommended to restore them to about in the culture conditions and medium30 minPost staining to avoid false positives.

6. To avoid cell loss during cell washing, a large one can be used during liquid aspirationTipWearing a small headbandTipHead sucking liquid.

7. The concentration of dye used is determined by specific experimental requirements.

8. Fluorescent dyes all have quenching issues. Please avoid light as much as possible during storage and use to slow down fluorescence quenching.

9. For your safety and health, please wear lab coats and disposable gloves when operating.