Technical Tips for working with Human Peripheral Blood Mononuclear Cell (PBMC) in ELISPOT Assays
- It is important to quick-spin vials before use to ensure content volumes.
- To maximize the use of each plate, an adhesive plate-sealing sheet has been included that can be adhered to the top of the plate to cover and protect unused wells that are intended to be used in subsequent assays. Use your thumbs to firmly adhere the sheet to the plate and use a razor blade to cut the sheet to expose only the necessary wells.
- We highly recommend the use of CTL Serum-free Media for freezing, washing, and testing PBMC. Even brief exposure to a mitogenic serum can cause high background while other sera can have suppressive effects. CTL also recommends using the CTL-LDCTM Kit for accurate live/dead cell counts.
- Deviations from specified temperatures, timing requirements, number of washing steps, and specified reagent preparation volumes may alter the performance of the assay.
- Plates can be washed manually or by a suitable automated plate washer with adjusted pin length and flow rate so membranes and spots are not damaged (CTL recommends BioTek ELx405).
- To avoid damage to the PVDF membrane in the wells, do not touch the membrane with pipette tips or with the plate washer. The PVDF membrane is permeable and protected by an underdrain. Avoid direct contact between the well bottom and wet surfaces, including paper towels or any other materials that can absorb liquid.
- While processing plates, the PVDF membrane at the bottom of the wells must remain wet.
- When underdrain and gloves are wet, the underdrain may be slippery and difficult to remove. Wipe gloves
and underdrain with paper towel before removing.
- After completion of the experiment, do not dry the ELISPOT assay plates at temperatures exceeding 37°C as this may cause the membrane to crack.
- Spots may not be readily visible while the membrane is still wet. Scan and count plates only after membranes have completely dried.
- Higher background appearing in the control wells can be potentially overcome by the following steps:
– When working with pre-cultured cells, wash the cells thoroughly in CTL-WashTM prior to the experiment
in order to avoid carryover of cytokines and other substances; use CTL-TestTM for testing PBMC.