Advanced Calcein AM Cell Viability Kit

This kit combines Calcein AM with 7-AAD to allow for easy and simultaneous labeling of live, membrane compromised, and dead cells within a single sample. Samples can be analyzed using a flow cytometer or fluorescent microscope.

Figure 1. Live cells with green fluorescence (right side of histogram) can be distinguished from dead cells (unstained, left side of histogram). Forward and side scatter graphs are also shown. Jurkat cells were stained with 1 µM Calcein AM and analyzed in a flow cytometer with a FL-1 99% attenuation filter. 93.0% of the live cells stained positive with Calcein AM (A, right), while 0.0% of ethanol-killed cells stained positive (B, right). Data courtesy of Dr. Kristi Strandberg, ICT 226:87-92.
Figure 2. Using 7-AAD, live cells (unstained, left side of each histogram) can easily be distinguished from dead/membrane compromised cells exhibiting red fluorescence (right side of each histogram). Jurkat cells were stained with 7-AAD, and analyzed using a flow cytometer in FL-3. Only 4% of untreated cells (A) are dead compared with 97.6% of ethanol-treated cells (B). Data courtesy of Dr. Kristi Strandberg, ICT 226:30-31.
Figure 3. Untreated Jurkat suspension cells were dually stained with 1 µM Calcein AM for 60 minutes at 37°C to detect live cells. Cells were then stained with 4 µM 7-AAD for 10 minutes on ice to detect membrane-compromised or dead cells. Panel A reveals green fluorescence-stained live cells. A few dead cells stained red due to 7-AAD uptake. Panel B shows a corresponding DIC image, which reveals cell morphology. Data courtesy of Dr. Kristi Strandberg (ICT 226:95).
Figure 4. Jurkat suspension cells were dually stained with 1 µM Calcein AM to detect live cells, and then stained with 4 µM 7-AAD to detect membrane-compromised or dead cells. Untreated live cells stained green (A). Occasional dead cells stained red due to 7-AAD uptake. Ethanol-killed cells all stained red due to 7-AAD uptake (B). A 50/50 mixture of the untreated and ethanol-killed cells shows both green and red cells (C). Data courtesy of Dr. Kristi Strandberg, ICT 226:95.
Figure 5. Jurkat cells were treated with DMSO (non-induced) or 1 µM staurosporine to induce apoptosis, then dually stained with 1 µM Calcein AM to detect live cells and 4 µM 7-AAD to detect membrane-compromised or dead cells. All of the non-induced cells fluoresced green only (A). Most of the induced cells were green (live), many cells were both green and red (compromised membranes, likely early apoptotic), and some were red (dead). Data courtesy of Dr. Kristi Strandberg, ICT 226:95.
Figure 6. Live cells fluoresce green due to esterase cleavage of Calcein AM, but do not fluoresce red as 7-AAD is excluded (LR, lower right). Live, membrane-compromised cells fluoresce green due to esterase cleavage of Calcein AM, and fluoresce red due to 7-AAD entry and DNA binding (UR, upper right). Dead cells fluoresce red due to 7-AAD entry, but lack active esterases and do NOT fluoresce green as Calcein AM is not cleaved (UL, upper left). Unstained cells, no fluorescence (LL, lower left).
Figure 7. Jurkat suspension cells were stained with Calcein AM, followed by 7-AAD, then analyzed in a flow cytometer with a FL-1 99% attenuation filter. Most live cells stained with Calcein AM (A; LR, lower right, 89.4%). No dead cells stained positive for Calcein (B; LR, lower right). Most dead cells stained positive for 7-AAD (B: UL, upper left, 98.9%). A mixture of live and ethanol-killed cells (C) shows both live (LR, 73.1%) and killed (UL, 23.0%) cells. Data of Dr. K. Strandberg.
Figure 8. Jurkat cells were treated with DMSO (A) or staurosporine (B), then stained with Calcein AM followed by 7-AAD. Calcein was analyzed on the 99% attenuated FL-1 channel, and 7-AAD on the FL-3 channel. Live cells fluoresce green (lower right of density plot A). Dually stained green and red fluorescing cells are membrane compromised (likely mid to late apoptotic) (upper right of density plot B). Necrotic cells fluoresce red (upper left, A and B). Data of Dr. Strandberg.
Figure 1. Live cells with green fluorescence (right side of histogram) can be distinguished from dead cells (unstained, left side of histogram). Forward and side scatter graphs are also shown. Jurkat cells were stained with 1 µM Calcein AM and analyzed in a flow cytometer with a FL-1 99% attenuation filter. 93.0% of the live cells stained positive with Calcein AM (A, right), while 0.0% of ethanol-killed cells stained positive (B, right). Data courtesy of Dr. Kristi Strandberg, ICT 226:87-92.
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Figure 1. Live cells with green fluorescence (right side of histogram) can be distinguished from dead cells (unstained, left side of histogram). Forward and side scatter graphs are also shown. Jurkat cells were stained with 1 µM Calcein AM and analyzed in a flow cytometer with a FL-1 99% attenuation filter. 93.0% of the live cells stained positive with Calcein AM (A, right), while 0.0% of ethanol-killed cells stained positive (B, right). Data courtesy of Dr. Kristi Strandberg, ICT 226:87-92.
Figure 2. Using 7-AAD, live cells (unstained, left side of each histogram) can easily be distinguished from dead/membrane compromised cells exhibiting red fluorescence (right side of each histogram). Jurkat cells were stained with 7-AAD, and analyzed using a flow cytometer in FL-3. Only 4% of untreated cells (A) are dead compared with 97.6% of ethanol-treated cells (B). Data courtesy of Dr. Kristi Strandberg, ICT 226:30-31.
Figure 3. Untreated Jurkat suspension cells were dually stained with 1 µM Calcein AM for 60 minutes at 37°C to detect live cells. Cells were then stained with 4 µM 7-AAD for 10 minutes on ice to detect membrane-compromised or dead cells. Panel A reveals green fluorescence-stained live cells. A few dead cells stained red due to 7-AAD uptake. Panel B shows a corresponding DIC image, which reveals cell morphology. Data courtesy of Dr. Kristi Strandberg (ICT 226:95).
Figure 4. Jurkat suspension cells were dually stained with 1 µM Calcein AM to detect live cells, and then stained with 4 µM 7-AAD to detect membrane-compromised or dead cells. Untreated live cells stained green (A). Occasional dead cells stained red due to 7-AAD uptake. Ethanol-killed cells all stained red due to 7-AAD uptake (B). A 50/50 mixture of the untreated and ethanol-killed cells shows both green and red cells (C). Data courtesy of Dr. Kristi Strandberg, ICT 226:95.
Figure 5. Jurkat cells were treated with DMSO (non-induced) or 1 µM staurosporine to induce apoptosis, then dually stained with 1 µM Calcein AM to detect live cells and 4 µM 7-AAD to detect membrane-compromised or dead cells. All of the non-induced cells fluoresced green only (A). Most of the induced cells were green (live), many cells were both green and red (compromised membranes, likely early apoptotic), and some were red (dead). Data courtesy of Dr. Kristi Strandberg, ICT 226:95.
Figure 6. Live cells fluoresce green due to esterase cleavage of Calcein AM, but do not fluoresce red as 7-AAD is excluded (LR, lower right). Live, membrane-compromised cells fluoresce green due to esterase cleavage of Calcein AM, and fluoresce red due to 7-AAD entry and DNA binding (UR, upper right). Dead cells fluoresce red due to 7-AAD entry, but lack active esterases and do NOT fluoresce green as Calcein AM is not cleaved (UL, upper left). Unstained cells, no fluorescence (LL, lower left).
Figure 7. Jurkat suspension cells were stained with Calcein AM, followed by 7-AAD, then analyzed in a flow cytometer with a FL-1 99% attenuation filter. Most live cells stained with Calcein AM (A; LR, lower right, 89.4%). No dead cells stained positive for Calcein (B; LR, lower right). Most dead cells stained positive for 7-AAD (B: UL, upper left, 98.9%). A mixture of live and ethanol-killed cells (C) shows both live (LR, 73.1%) and killed (UL, 23.0%) cells. Data of Dr. K. Strandberg.
Figure 8. Jurkat cells were treated with DMSO (A) or staurosporine (B), then stained with Calcein AM followed by 7-AAD. Calcein was analyzed on the 99% attenuated FL-1 channel, and 7-AAD on the FL-3 channel. Live cells fluoresce green (lower right of density plot A). Dually stained green and red fluorescing cells are membrane compromised (likely mid to late apoptotic) (upper right of density plot B). Necrotic cells fluoresce red (upper left, A and B). Data of Dr. Strandberg.

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Product Manual
Safety Data Sheet

SKU: 9154

Size: 25 - 250 Tests
Price:
$299.00
Ships: 1-2 business days
  1. Prepare samples and controls.
  2. Dilute 10X Cellular Assay Buffer 1:10 with diH2O.
  3. Reconstitute Calcein AM with 50 µL DMSO.
  4. Dilute 2 mM Calcein AM stock solution 1:5 by adding 200 µL diH2O or PBS, forming a 400 µM stock solution.
  5. Stain with Calcein AM at a concentration between 1-10 µM. The ideal staining concentration can vary based on cell line, application, etc., and should be determined by the end user. The recommended sample size is 0.4 mL.
  6. Add Calcein AM to each sample and mix gently. For example, to stain a 0.4 mL sized sample with 10 µM Calcein AM, add 10 µL of the 400 µM stock solution.
  7. Incubate approximately 1 hour.
  8. Reconstitute 7-AAD with 260 µL DMSO, forming a 200X stock concentrate.
  9. Add 7-AAD to each sample at 1:200 (e.g., add 2 µL to 400 µL of sample).
  10. Analzye with a flow cytometer or fluorescence microscope. Calcein AM excites at 494 nm and emits at 520 nm. 7-AAD excites at 546 nm and emits at 647 nm.
  • Calcein AM Reagent, 1 vial, #6696
  • 7-Aminoactinomycin D (7-AAD) vital dye, 0.26 mg, 2 vials, #6163
  • Hoechst 33342, 1 vial, #639
  • 10X Cellular Assay Buffer, 60 mL, 1 bottle, #6695
  • Kit Manual

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