| BACKGROUND/TECHNICAL INFORMATION |
Swiss-Prot link P14222: Perforin 1 (human) (precursor)
General Note:
Both primary and transformed lines are permeabilized by the perforin, but the concentration required to produce 50% permeabilized cells varies substantially according to cell type (e.g. lymphoid, epithelial, mesenchymal), sensitivity of the cell line and whether cells are suspended or adherent. Generally, consumption of perforin is greater in the testing of adherent cells due to non-specific binding of the pore-forming protein to the microwells. Perforin should be pre-titrated against the desired cell line to identify the minimal permeabilizing concentration for protein delivery (see below). This concentration will allow effective delivery of the protein without producing excessive background necrosis and represent the nanograms that achieve less than 10-20% permeabilization of the target cell in question.
Supplied with Perforin Dilution Buffer (1ml).
Protocol for Intracellular Perforin Mediated Protein Delivery a. Materials Needed:
- Microfuge tubes (1.5ml)
- Calcium stock solution: 100mM CaCl2 in water.
- Ca2+-HEPES buffer: 20mM HEPES containing 150mM NaCl and 2.5mM CaCl2, pH 7.4
- BSA-HEPES buffer: 1% BSA solubilized in HEPES buffer (without Ca2+).
- Fatty acid-free BSA.
- Perforin Dilution Buffer (included). b. Pre-titration of PFN against Desired Cells:
Immediately before the planned experiment, perforin should be titrated to identify concentration that minimally permeabilizes targets. Incubate desired cells with increasing concentrations of perforin for 15 min., then determine percentage of cells that have undergone permeabilization by Trypan Blue or Propidium Iodide stain.
1) Wash cells twice in Ca2+-HEPES buffer to remove serum from cell mixture and re-suspend at density
of 1x106 per 50µl in same buffer.
2) For each sample, dilute perforin to twice the desired final concentration in 50µl of HEPES/1% BSA
buffer to microfuge tubes.
3) Add 50µl of media control or dilute perforin to 50µl of cells in microfuge tubes.
4) Incubate at 37°C for 15 min.; periodic mixing is not required.
5) Perform readout to assess permeabilization.
d. Experimental Protocol:
1) Wash cells twice in Ca2+-HEPES buffer to remove serum from cell mixture and re-suspend at density
of 1x106 per 50µl in same buffer.
2) Add Granzyme B (Prod. No. ALX-200-602) (1µg/ml); generally 1-2µl of stock is sufficient.
3) For desired samples, dilute perforin to twice the desired final concentration in 50µl of HEPES/1% BSA
buffer to microfuge tubes.
4) Add 50µl of media control or dilute perforin to 50µl of cells in microfuge tubes.
5) Incubate at 37°C for desired time; periodic mixing is not required.
6) After incubation perform readout to assess permeabilization.
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