TRITC tagging of Fetal Bovine Serum, TECAN v ImageJ measurements

pnc-Si chips prewetted with PBS then incubated with fetal bovine serum (unknown molarity) for 2 hours under refrigerated conditions. Thoroughly rinsed with reaction buffer (perfused 5 times). TRITC (2 mg/mL) added to dry chips and incubated at room temperature for one hour. Then rinsed once with reaction buffer then rinsed thoroughly with distilled de-ionized (DDI) water.

Chips Placed in a black 96-well plate and read with the TECAN (excitation wavelength 557 nm, whatever wavelength 576 nm with a gain of 40%). Four chips were incubated with serum, one was incubated with reaction buffer, one chip was dry and untreated.

These are the green bars in Figure 1. Values for the incubated chips are typically ~8200 though one was as low as 5151. The wetted chip was the highest reading (8513), the dry chip was equal to the incubated chips (8228). Interestingly, while the empty wells of the black plate were around 1000, three wells with DDI water read closer to 300.

The ImageJ numbers were all between 55.5 to 66.856. Which is generally about the value I see for non-incubated surfaces.

I did another chip, with the same protocol except I incubated in the serum overnight in the refrigerator. I did not use the TECAN, just the fluorescence scope. Unfortunately I had the same result, with readings averaging 62. There does seem to be some texture to the surface of the chips, so I assume there is a layer of serum on the chip, but that the TRITC is not attaching to the serum. I’ll try more time with the TRITC.

A third attempt.  Both serum and TRITC incubations were performed at overnight at 4ºC. Here is the full protocol.

• Incubate chips with pure serum (from stock of serum – in -20°C freezer somewhere) overnight at 4ºC
• Make 100 ml of reaction buffer: 100 mM Sodium carbonate/bicarbonate adjusted to pH 9.0
• Dissolve 5 mg TRITC in reaction buffer at 2 mg/ml.
• After chip incubation with serum,  rinse profusely with reaction buffer.
• Soak chip in TRITC reaction solution overnight at 4°C. Keep in the dark (covered under aluminum foil).
• Rise chip profusely to remove unreacted dye with DDI water.
• Inspect