What are the activation effects and compatibility of anti-CD3/CD28 functional antibody coated plates with consumables?

The 48-well delivery period should be asked in advance. Domestic functional anti-reflection goods may take 1-3 days, imported small packages cold chain is usually 1-2 weeks, pre-packed sheets will be customized for a longer time, so do not wait until the experiment before ordering. In terms of operation, I am used to using two 24-well plates, 1 mL system per well, and the cell density is about 1 × 10 ^ 6/mL. After the antibody is matched with sterile PBS, filter or pay attention to aseptic operation. Coat it at 4 ℃ overnight, suck it out before use, and wash it with PBS once. If you want to improve efficiency, you can fix the volume of the coating liquid and the order of sample addition to reduce human error. I usually observe whether the cells are aggregated for 24 hours, watch CD25/CD69 for 48 hours, and count for 72 hours to see the amplification factor. If you want to control the cost, you can test whether the antibody can be recycled once or twice, but you must confirm the sterility and activity, and it is not recommended to use it repeatedly for a long time. The single-hole budget self-packing coating model can usually be pressed lower than the pre-packed coating board, but stability and personnel time should be considered together. Do not rework the whole batch of experiments in order to save antibody money.

I have used both self wrapped and pre wrapped bedding boards. The advantage of self coating is that the antibody concentration, ratio, and buffer solution can be adjusted, which is suitable for early optimization; The downside is that it takes up time each time and the board gap needs to be controlled by oneself. Pre packaged boards can be used immediately upon opening the box, with generally smaller batch differences but higher unit prices and longer customized delivery times. 48 wells at once, if self coated, the total antibody per well is 0.5-1 μ g, and the 48 wells are approximately 25-50 μ g. It is more cost-effective to buy 100 μ g specifications for several tests; Pre packaged boards are priced according to the board, and the cost per hole is usually higher than that of self packaged boards, but it saves labor and failure risks. Suggest using the same donor PBMC as a parallel control: the same batch of antibodies, half self coated with regular plates and half pre coated with plates, compare the number of CD25/CD69 positive cells at 48 hours and cells at 72 hours. Cost accounting includes antibodies, boards, cleaning solutions, and labor, not just the unit price of the boards. It is difficult to give an absolute budget, so it is recommended to consult three companies first and compare them based on the actual antibody dosage per well.

There are indeed differences between ordinary TC treatment boards and imported high adsorption boards, but ordinary TC boards are not unusable. I have made a comparison. The ordinary TC plate is directly coated with anti-CD3/CD28, and the CV between holes will be larger than the high adsorption plate, especially the edge holes. It is recommended to fix a plate number for pre-testing. After coating, use fluorescent secondary antibody or HRP secondary antibody to measure the uniformity of adsorption between wells, and then decide whether it is worth changing the plate. Coating conditions I usually use sterile PBS dilution, anti-CD3 and anti-CD28 1-5 μg/mL, 0.5 mL per well, 4 ℃ overnight; before use, suck off the coating solution, wash once with PBS, and then block with 2% BSA or medium for 30-60 minutes. The surface of the TC treatment plate is hydrophilic, and antibody adsorption is OK, but do not stack it to avoid uneven temperature. Imported plates such as high adsorption plates are indeed more stable, but the cost of a single hole will go up, and the size of 48 holes should be calculated as the total consumption account.