Total Cytotoxicity Assay

1
Total Cytotoxicity Assay

• Immunochemistry Technologies, LLC
• www.immunochemistry.com
• 9401 James Avenue South, Suite 155
• Bloomington, MN 55431
• 800-829-3194, 952-888-8788
• fax [phone]
• Email gary_at_immunochemistry.com

Catalog 971 125 tests 289.00
Catalog 972 250 tests 479.00
2

• When cells go through apoptosis, they package
  their material. This inhibits the release of
  51Cr, LDH, other dyes, and the detection of vital
  stains.
• These older assays underestimate the levels of
  cytotoxicity.

3

• ICTs total cytotoxicity assay uses a viability
  stain in conjunction with a cell-permeant poly
  caspases reagent to concurrently label dead and
  apoptotic cells.
• This new assay provides an accurate measurement
  of cytotoxicity without using radioactive
  materials.

4
Total Cytotoxicity Assay

• Easy - run samples in a single tube
• Safe no radioactivity
• Fast - finish in one day
• Sensitive - measure killed and apoptotic cells
• Accurate analyze cells with a flow cytometer

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Kit Reagents

• CFSE
• 7-AAD
• Poly Caspases Detection Reagent

125 tests 289.00 Catalog 971 250 tests
479.00 Catalog 972
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Assay Overview
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Add poly caspases reagent
Add effector cells (PBMC, CD8, NK cells, etc.)
Reagent will penetrate through cells and bind to
active caspases
Effector cells will act on target cells
Following incubation, add Live/dead stain. This
will stain cells in necrosis and late apoptosis
Stain target cells with membrane stain
Purify target cells (K562, monocytes, etc.)
If cells are not going through apoptosis, reagent
will leave the cells
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Draw a gate around the target cells
Create a live/dead vs. caspase graph
7AAD- VAD population is not detectable by PI
staining alone, nor will this population allow
for the detection of death by other assays.
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Apoptotic Cells
12.51 251
501
1001 E/T Ratios
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Examples

• Gamma delta lymphocytes activated with BCG
  vaccination. Olin et al., Journal of
  Immunological Methods 297, 1-11.

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Natural Killer activity against K562 cells
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Antigen directed cytolytic activity against M.
bovis infected monocytes
25
P0.04
20
15
Cytolytic Activity
10
5
0
Non-vaccinated BCG-vaccinated

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?d vs. CD8 T lymphocytes antigen driven
cytolytic activity
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Gamma Delta lymphocyte subsets

• CD4, CD8, or CD4 and CD8 lymphocytes were
  depleted prior to gamma delta purification.
  Publication in preparation

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Cytolytic activity
Dual Positive Cytotoxicity
40
35
30
P0.03
25
Cytotoxicity Activity
P0.025
20
15
10
5
0
gd/CD4-CD8-
gd/CD4-CD8
gd/CD4CD8-
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Morphines effect on Gamma Delta lymphocytes

• Morphine was administered in-vivo prior to BCG
  vaccination Submitted to Journal of
  Neuroimmunology.

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Natural Killer activity
BCG
BCG-Morphine
Control
To-pro-3
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Innate activity
P0.02
Cytolytic activity
Non-morphine
Morphine
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Gamma Delta cytolytic activity agains M. bovis
infected CNS cells

• Submitted for publication, Infection and immunity.

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In-vitro model

• Microglia isolated from 1 day old piglets
• gd lymphocytes from non-vaccinated and BCG
  vaccinated animals

Non-vaccinated animals BCG
vaccinated animals
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Cytolytic activity
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Total Cytotoxicity Assay

• Easy - run samples in a single tube
• Safe no radioactivity
• Fast - finish in one day
• Sensitive - measure killed and apoptotic cells
• Accurate analyze cells with a flow cytometer

23
Call now to order1-800-829-3194
Catalog 971 125 tests 289.00
Catalog 972 250 tests 479.00

• Immunochemistry Technologies, LLC
• www.immunochemistry.com
• 9401 James Avenue South, Suite 155
• Bloomington, MN 55431
• 952-888-8788 fax [phone]
• Email gary_at_immunochemistry.com