LIVE／DEAD?FixableDeadCellStainKits

實驗概要

The LIVE/DEAD?  Fixable Dead Cell Stain Kits use a novel method to evaluate the  viability of mammalian cells by flow cytometry. These assays are based  on the reaction of a fluorescent reactive dye with cellular amines. The  reactive dye can permeate the compromised membranes of necrotic cells  and react with free amines both in the interior and on the cell surface,  resulting in intense fluorescent staining. In contrast, only the  cell-surface amines of viable cells are available to react with the dye,  resulting in relatively dim staining (Figure 1). The difference in  intensity between the live and dead cell populations is typically  greater than 50-fold (Figure 2). The discrimination is completely  preserved following fixation of the sample by formaldehyde, under  conditions that inactivate pathogens. Moreover, these single-color  assays use only one channel of a flow cytometer, leaving the other  channels available for multicolor experiments. The assays can also be  used to detect dead cells by microscopy; however, the difference in  fluorescence intensity of the live and dead cells can be appreciable,  making it relatively difficult to simultaneously photograph the two  populations.

The single-color LIVE/DEAD? Fixable Dead Cell Stain Kits are  identical except for the fluorescent color of the included dye—blue,  violet, aqua, yellow, green, red, far red, or near?IR (infra red). Cells  labeled by green fluorescent or red fluorescent reactive dye (L23101  and L23102, respectively) are excited by the 488 nm line of an argon-ion  laser; green fluorescence is typically detected in the green channel of  the flow cytometer (530/30 nm) and red fluorescence is detected in the  red channel (630/30 nm). The blue fluorescent reactive dye (L23105)  requires UV (350–360 nm) excitation with fluorescence emission read at  ~450 nm. The violet fluorescent reactive dye (L34955) requires violet  (~405 nm) excitation with fluorescence emission read at ~440 nm. The  aqua fluorescent reactive dye (L34957) is efficiently excited with ~405  nm light (or UV light) and has fluorescence emission monitored at ~525  nm. The yellow fluorescent reactive dye (L34959) requires violet (~405  nm) excitation with fluorescence emission read at ~575 nm (appropriate  channels for the violet-excitable reactive dyes may vary depending on  the instrument). The far red (L10120) and near-IR (L10119) fluorescent  reactive dyes are excited at 633/635 nm with fluorescence emission  monitored at 665 nm and 775 nm, respectively.

The LIVE/DEAD? Fixable Dead Cell Stain Sampler Kit (L34960) contains  one vial of each of the eight different fluorescent reactive dyes.  Invitrogen also offers a LIVE/DEAD? Reduced Biohazard Cell Viability Kit  (L7013) for determining cell viability. It is based on a different  staining principle using two dyes of different colors, and is described  separately.

Table 1. Approximate fluorescence excitation and emission maxima for the LIVE/DEAD? Fixable Dead Cell Stain single-color dyes.

*Approximate fluorescence excitation (Ex) and emission (Em) maxima, in nm.


 
 

主要試劑

Table 2. Contents and storage information.

Number of assays: Each individual kit provides sufficient material  for approximately 200 flow cytometry assays, 40 assays per vial of the  reactive dye. The Sampler Kit contains one vial of each fluorescent  reactive dye with 40 assays per vial of the reactive dye, for a total of  320 assays per kit.

Approximate fluorescence excitation/emission maxima: See Table 2.
Before You Begin

Allow the reagents to warm to room temperature before opening the vials.

Materials Required but Not Provided

• Phosphate buffered saline (PBS)

• PBS with 1% bovine serum albumin (BSA)

• Formaldehyde

• AbC? Anti-Mouse Bead Kit (if calculating compensation in multicolor immunophenotyping experiments using mouse antibodies)

• ArC?  Amine Reactive Compensation Bead Kit (if calculating compensation in  multicolor experiments using a LIVE/DEAD? Fixable Dead Cell Stain)

Caution: DMSO  is hazardous; avoid contact with skin and eyes and do not swallow.  Handle reagents containing DMSO using equipment and practices  appropriate for the hazards posed by such materials.

實驗步驟

The following  protocol has been used successfully in our laboratories to  differentially stain live and dead cells and then to fix the cells in  formaldehyde for subsequent analysis by flow cytometry. Excellent  results have been obtained with a variety of cell types, including:  Jurkat, MDCK, COLO205, CHO-K1, K-562, HeLa, P3X, 3T3, B35, BPAC, CHO-M1,  A549, MMM, MRC5, U205, U266, 293 MSR, and RAW tissue-culture cell lines  and human peripheral blood lymphocytes. If another staining reaction is  to be performed on the same sample, determine the optimal staining  sequence for the two procedures and whether or not the additional  staining reaction will tolerate fixation by formaldehyde.

1. Dye Preparation

For convenience, the reactive dye in each individual kit is supplied  in five separate vials, while the sampler kit contains one vial of each  reactive dye. Each vial provides sufficient material for staining at  least 40 cell samples. However, once reconstituted, the DMSO solution of  reactive dye is somewhat unstable, especially if exposed to moisture.  Unused portions may be used for up to 2 weeks if stored at ≤–20°C,  protected from light and moisture.

1)       Bring one vial of the fluorescent reactive dye (Component A) and the  vial of anhydrous DMSO (Component B) to room temperature before removing  the caps.

2)      Add 50 μL of DMSO to the vial of reactive dye. Mix well and visually confirm that all of the dye has dissolved.

3)      Use the solution of reactive dye as soon as possible (see below), ideally within a few hours of of reconstitution

2. Staining the Cells

Buffers appropriate for cell staining include phosphate-buffered  saline (PBS), Hanks’ Balanced Salt Solution (HBSS), and Dulbecco’s PBS  (D-PBS), without extraneous proteins such as bovine serum albumin or  serum. When using an amino-reactive dye, for example the violet  fluorescent reactive dye (Cat. no. L34955), Tris buffers and solutions  containing sodium azide or extraneous protein should not be used for  cell resuspension and washing.

1)      Wash the cells once with 1 mL of PBS.

2)      Resuspend the cells in 1 mL of PBS.

3)      Count the cells and adjust the density with PBS to 1 × 10⁶ cells in a 1 mL volume.

4)      Add 1 μL of the reconstituted fluorescent reactive dye (from step 1.3) to 1 mL of the cell suspension and mix well.

5)      Incubate at room temperature or on ice for 30 minutes, protected from light.

6)      Wash the cells once with 1 mL of PBS, and resuspend the cells in 900 μL of PBS.

Note: If  fixation is not required, then you can skip the steps 2.7–2.10 below.  Instead, wash the cells twice with 1 mL of PBS with 1% bovine serum  albumin, and resupend in 1 mL of PBS with 1% bovine serum albumin.

7)      Add 100 μL of 37% formaldehyde.

8)      Incubate at room temperature for 15 minutes.

9)       Wash once with 1 mL of PBS with 1% bovine serum albumin, and resuspend  the cells in 1 mL of PBS with 1% bovine serum albumin.

10)   Analyze the fixed cell suspension by flow cytometry using the  appropriate excitation and detection channel (these may vary depending  on instrument used):

• Blue fluorescent reactive dye uses UV excitation and ~450 nm emission (450/50 nm or other filter)

• Violet fluorescent reactive dye uses 405 nm excitation and ~450 nm emission (450/50 nm or other filter)

• Aqua fluorescent reactive dye uses 405 nm excitation and ~525 nm emission (525/50 nm or other filter)

• Yellow fluorescent reactive dye uses 405 nm excitation and ~575 nm emission (575/26 nm, 585/42 nm, or similar filter)

• Green fluorescent reactive dye uses 488 nm excitation and ~530 nm emission (530/30 nm or other filter)

• Red fluorescent reactive dye uses 488 nm excitation and ~ 585 nm emission (585/42 nm, 610/20 nm, or other filter)

• Far red fluorescent reactive dye uses 633/635 nm excitation and ~660 nm emission

• Near-IR fluorescent reactive dye uses 633/635 nm excitation and ~780 nm emission

3. Intracellular Staining

This procedure is completely compatible with common fixation and  permeabilization methods for performing intracellular staining for flow  cytometry. The procedure for staining with a LIVE/DEAD? Fixable Dead  Cell Stain combined with immunophenotyping is as follows:

1)      Centrifuge a sample of cells in suspension containing at least 1 × 10⁶ cells. Discard the supernatant.

2)      Wash the cells once with 1 mL of PBS.

3)      Resuspend the cells in 1 mL of PBS.

4)      Count the cells and adjust the density with PBS to 1 × 10⁶ cells in a 1 mL volume.

5)      Add 1 mL of the reconstituted fluorescent reactive dye (from step 1.3) to 1 mL of the cell suspension and mix well.

6)      Incubate at room temperature or on ice for 30 minutes, protected from light.

7)      Wash the cells with 1 mL of PBS and resuspend in 100 μl of PBS.

8)      Stain as usual for surface markers, and incubate for desired time for antibody staining.

9)      Wash the cells with PBS and resuspend in 900 μl of PBS

10)  Add 100 μl of 37% formaldehyde.

11)  Incubate at room temperature for 15 minutes.

12)   Wash once with 1 mL of PBS with 1% bovine serum albumin, and resuspend  the cells in 100 μl of PBS with 1% bovine serum albumin.

13)  Add permeabilization reagent and stain as usual for intracellular markers. Incubate for desired time for antibody staining.

14)   Wash once with 1 mL of PBS with 1% bovine serum albumin, and resuspend  the cells in 1 mL of PBS with 1% bovine serum albumin.

15)  Analyze the fixed cell suspension by flow cytometry using the appropriate excitation and detection channel (from step 2.11)

4. Compensation Using ArC? Amine Reactive Beads

The ArC? Amine Reactive Compensation Bead Kit is designed to  facilitate compensation when using any of the LIVE/DEAD? fixable dead  cell stains, providing a consistent, accurate and simple-to-use  technique for the setting of flow cytometry compensation. The ArC? Amine  Reactive Compensation Bead Kit includes two types of specially modified  polystyrene microspheres to allow easy compensation of the LIVE/DEAD?  fixable stains: the ArC? reactive beads (Component A), which bind any of  the amine-reactive dyes, and the ArC? negative beads (Component B),  which have no reactivity. After incubation with any amine reactive dye,  the two kit components will provide distinct positive and negative  populations of beads that can be used to set compensation. We recommend  the following protocol for using ArC? Amine Reactive Compensation Bead  Kit for compensation:

1)      Gently vortex ArC? Amine Reactive Compensation Bead Kit components for 30 seconds to completely resuspend before use.

2)      Add 1 drop of ArC? reactive beads (Component A) to a labeled sample tube.

3)      Allow ArC? reactive beads to sit in the tube for 5 minutes to warm to room temperature.

4)       Prepare fluorescent amine-reactive dye according to instructions  included in the LIVE/DEAD? Fixable Dead Cell Kit. For optimal  performance of the ArC? reactive beads, use freshly prepared  amine-reactive dye. Do not use previously frozen dye solution.

5)       Add the amount of LIVE/DEAD? fixable dead cell stain listed in Table 3  to the bead suspension and mix well. Make sure to deposit the  amine-reactive dye directly to the bead suspension.

Table 3. Amount of amine-reactive LIVE/DEAD? fixable dead cell stain for use with ArC? reactive beads

      6)      Incubate for 30 minutes at room temperature, protected from light.

7)      Add 3 mL of PBS or other buffer to sample tube. Centrifuge for 5 minutes at 300 × g.

8)       Carefully remove all the supernatant from tube. If using the red  fluorescent reactive dye (Cat. no. L23102), repeat step 4.7.

9)      Resuspend bead pellet by adding 0.5 mL of buffer to sample tube

10)  Add one drop of ArC? negative beads (Component B) to sample tube. Mix thoroughly.

11)  Vortex tubes before analyzing using flow cytometry.

12)   Perform manual or automatic compensation according to the preferred  procedure for the flow cytometer in use. Gate on the bead singlet  population based on FSC and SSC characteristics.

5. Combining ArC? and AbC? Kits

The AbC? Anti-Mouse Bead Kit provides a consistent, accurate, and  simple-to-use technique for the setting of flow cytometry compensation  when using fluorochrome-conjugated mouse antibodies. The kit contains  two types of specially modified polystyrene microspheres: the AbC?  capture beads (Component A) that bind all isotypes of mouse  immunoglobulin, and the negative beads (Component B) that have no  antibody binding capacity. After incubating with a  fluorochrome-conjugated mouse antibody, the two components provide  distinct positive and negative populations of beads that you can use to  set compensation. You can use the AbC? Anti-Mouse Bead Kit and the ArC?  Amine Reactive Compensation Bead Kit together to calculate compensation  in multicolor immunophenotyping experiments that incorporate a  LIVE/DEAD? fixable dye by following the protocol outlined below:

1)       Gently vortex the ArC? Amine Reactive Compensation Bead Kit and the  AbC? Anti-Mouse Bead Kit components for 30 seconds to completely  resuspend before use.

2)       Label a sample tube for the amine-reactive dye you are using and add 1  drop of ArC? reactive beads (Component A in the ArC? Amine Reactive  Compensation Bead Kit) to the labeled sample tube. Allow ArC? reactive  beads to sit in the tube for 5 minutes to warm to room temperature.

3)       Prepare fluorescent reactive dye according to kit instructions included  in the LIVE/DEAD? Fixable Dead Cell Stain Kit. For optimal performance  of ArC? reactive beads, always use freshly prepared amine-reactive dye.  Do not use previously frozen dye solution

4)       Add the amount of LIVE/DEAD? fixable dead cell stain listed in Table 3  to the bead suspension and mix well. Make sure to deposit the  amine-reactive dye directly to the bead suspension.

5)       Label another sample tube for each fluorochrome-conjugated antibody you  are using, and add 1 drop of AbC? capture beads (Component A in the  AbC? Anti-Mouse Bead Kit) to each labeled tube.

6)       Add a pre-titrated amount of antibody conjugate to the appropriate tube  and mix well. Make sure to deposit the antibody directly to the bead  suspension

7)      Incubate for 30 minutes at room temperature, protected from light

8)      Add 3 mL of PBS or other buffer to each sample tube. Centrifuge at 300 × g for 5 minutes to collect beads.

9)       Carefully remove all supernatant from each tube. If using the red  fluorescent reactive dye (Cat. no. L23102), repeat step 5.8 for that  tube.

      10)  Resuspend bead pellet by adding 0.5 mL of staining buffer to each sample tube.

11)   Add one drop of negative beads (Component B in the AbC? Anti-Mouse Bead  Kit) to sample tube(s) containing the AbC? capture beads.

12)   Add one drop of ArC? negative beads (Component B in the ArC? Amine  Reactive Compensation Bead Kit) to sample tube(s) containing the ArC?  reactive beads.

13)  Vortex tubes before analyzing using flow cytometry.

14)   Perform manual or automatic compensation according to the preferred  procedure for the flow cytometer in use. Gate on the bead singlet  population based on FSC and SSC characteristics.

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