Table 1.

Conventional and the covariance-based α-factors

FRET pairs				Labeling ratio		α-factors			FRET efficiencies (%)
Donor: Alexa-Fluor 546		Acceptor: Alexa-Fluor 647				Spectral	Covariance-based		Quenching	Quenching and sensitized emission
mAb	Epitope	mAb	Epitope	Ld	La	α0a	αb	αcubicc	Qd	E0e	Ef
A
L368	β2m	W6/32	MHCI heavy-chain	4.7	1.7	10.07	0.35	—	28	2	30
L368		W6/32 lowg				9.78	0.40	—	19	2	21
L368 lowg		W6/32				9.88	0.29	—	26	1	26
B
W6/32	MHCI heavy-chain	L368	β2m	1.5	2.1	0.13	0.14	—	19	26	25
W6/32		L368 lowg				0.12	0.09	—	16	18	22
W6/32 lowg		L368				0.13	0.16	—	23	27	24
C
L368	β2m	L368	β2m	4.7	2.1	3.96	0.16	0.21	39	1	13h
W6/32	MHCI heavy-chain	W6/32	MHCI heavy-chain	1.5	1.7	0.33	0.10	0.14	40	6	11h

Deduced FRET efficiencies measured between the β₂m (light-chain) and heavy-chain subunits of the MHCI receptor as well as between its heavy-chain subunits on the surface of FT T-lymphoblast cells by using Alexa-Fluor 546- and Alexa-Fluor 647-conjugated mAbs.

^aThe conventional (or spectral) α-factors (α₀) have been calculated according to Eq. 2 of the main text by using the mean intensities of the samples labeled with the donor and the acceptor as well as the labeling ratios and absorption coefficients. Because of the 1:1 stoichiometry of the two subunits of the same MHCI molecule, unity was used for the ratio of the labeled receptors (B_d/B_a). All data in this table are representative ones of three different measurements giving similar results, with relative errors <15% (mean ± SE).

^bCovariance-based α-factor at the donor side (α) was determined as the mean value of the corresponding cell-by-cell distribution of α obtained as the positive root of the quadratic polynomial in Eq. 9 written for the cell-by-cell distributions of the D, p, and q coefficients, examples of which are shown in Fig. 2. In calculation of the D coefficient in Eq. 9, for the noncompeting case of FRET measurement between the β₂m and heavy-chain subunits, the d value of the FRET sample was approximated by the mean of d₀ distribution of the corresponding single-donor labeled sample.

^cIn the case of FRET indicating homo-association between the MHCI receptors, instead of using d₀ of the single-donor labeled sample, the d′ value of the FRET sample has been corrected according to Eq. 19 and the positive root of the cubic polynomial in Eq. 20. This resulted in a meaningful FRET efficiency (α_cubic) that was used in the calculation of E. Whereas the root of the quadratic polynomial of Eq. 9 has been found for each cell and the cell-by-cell distribution of α has been determined, this latter calculation have been carried out only with mean values.

^dQuenching efficiency (Q) is defined as the relative change in the I₁ donor fluorescence due to the mAb used as acceptor. Mean values of the corresponding cell-by-cell distributions, defined as Q = 1−I₁/I_1,d where I₁ is intensity of the double-labeled sample and I_1,d is the mean intensity of the sample labeled only with the donor, are listed. In the case of competing mAbs for the measurement of MHCI homo-association, it also contains the intensity-reducing effect of mAb competition and the effect of FRET.

^eE₀ has been calculated as the mean of the corresponding cell-by-cell distribution obtained from the A′ distribution by using E₀ = A′/(α₀ + A′) (see Eq. S12 in the Supporting Material) with the conventional α-factor (α₀) as an input constant.

^fE has been calculated as the mean of the corresponding cell-by-cell distribution obtained from the A′ distribution by using E = A′/(α + A′) with the covariance-based α-factor (α) as an input constant.

^gIn this case, “low” means one-half of the saturating amount of the mAb concentration. These values were calculated by using α_cubic, the solution of Eq. 20 with m = 3.25, b = 0.17 (R² = 0.86) obtained by fitting the corresponding Q−Q′ plot like that shown in Fig. 3A.