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For pipetting the cells, tips with wide openings are preferred to reduce shear forces that induce apoptosis in T cells. (Avoid touching the membrane with the pipet tips).
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T cells can actively migrate during the assay’s duration. In particular, T cells that have been preactivated in vivo or in vitro can migrate rapidly, leaving a cytokine trail. Also, if the plates are shaken (due to banging of the incubator door, for example) or moved during the capture period, the cells will roll, leaving streaks and irregularly shaped spots.
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To ensure equal heat distribution, stacking the plates is not recommended.
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The best way is to put the plates upright into the flow hood over night. Make sure not to dry them at higher temperatures than 37 °C, as the membranes might crack.
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If kept protected from light, red plates are stable for up to five months. Blue assay plates are more stable, but it would be advisable to keep them protected from light as well.
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Yes, T cells can be frozen and used for ELISPOT. If CTL protocols are followed and our freezing media are used, human PBMC samples can be frozen without any loss in functionality.
In addition, CTL offers a wide range of cryopreserved PBMC samples, both characterized and uncharacterized. Please contact us for more information.
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Yes, CTL provides protocols for single- and double-color ELISPOT assays. CTL also offers wet laboratory training and assay consultation. Please inquire at info@immunospot.com for details.
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Yes, CTL can analyze double color wells, including those with double positive spots using the ImmunoSpot® dual-color software feature.
What kind of pipet tips can I use in ELISPOT assays?