Intracellular distribution of lipophilic fluorescent probes in mammalian cells.

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and 2-p-toluidinyl-6-naphthalene sulfonate (TNS) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.     

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes

EL4 cells were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membrane phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the $\text{P}$ value of both probes compared to the $\text{P}$ value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, $\text{P}$ values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in $\text{P}$ values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization for monitoring physical properties of membranes (such as ‘fluidity’) is discussed.     

The study of rous sarcoma virus-transformed baby hamster kidney cells using fluorescent probes

The fluorescent probes pyrene, pyrene butyric acid and $\text{N-}\text{phenyl}$ 1-naphthylamine have been used to investigate the changes that accompany in vitro transformation of a baby hamster kidney cell line using Rous sarcoma virus. The fluorescent probes which reside in the membrane were used to compare the changes in microviscosity and polarity of the membranes of normal cells with two transformed cell lines. The spectrofluorimetric data indicate that following transformation the probe $\text{N-}\text{phenyl}$ 1-naphthylamine resides in a more polar environment. However, using the probe pyrene, the yield of excimer indicates decreased mobility of this probe in the membrane of transformed cells. The data also indicate differences between the two transformed cell lines. Laser photolysis was used to study the lifetime of the pyrne probes and the quenching of the pyrene fluorescence in the membrane by several different quenching molecules. The data indicate differences between the three cell lines and suggest that transformation decreases movement within the membrane.     

Resolution of plasma membrane lipid fluidity in intact cells labelled with diphenylhexatriene

The partitioning of fluorescence probes into intracellular organelles poses a major problem when fluorescence methods are applied to evaluate the fluidity properties of cell plasma membranes with intact cells. This work describes a method for resolution of fluidity parameters of the plasma membrane in intact cells labelled with the fluorescence polarization probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The method is based on selective quenching, by nonradiative energy transfer, of the fluorescence emitted from the plasma membrane after tagging the cell with a suitable membrane impermeable electron acceptor. Such selective quenching is obtained by chemical binding of 2,4,6-trinitrobenzene sulfonate (TNBS), or by incorporation of $\text{N-}\text{bixinoyl}$ glucosamine (BGA) to DPH-labelled cells. The procedures for determination of lipid fluidity in plasma membranes of intact cells by this method are simple and straightforward.     

Lipid dynamics and protein-lipid interactions in rat colonic epithelial cell basolateral membranes

Lipid dynamics and lipid-protein interactions were examined in basolateral membranes prepared from rat proximal and distal colonic epithelial cells. The results demonstrate that: (1) these membranes have a high lipid fluidity, as assessed by steady-state fluorescence polarization studies using seven fluorescent probes; (2) lipid compositional differences exist between these membranes but their fluidity is similar; (3) fluorescence polarizations studies, using diphenylhexatriene (DPH), detect a thermotropic transition at 22–23°C in each membrane; (4) several membrane protein activities, including adenylate cyclase and sodium-potassium dependent adenosine triphosphatase ($\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$) appear to be functionally dependent on the physical state of the proximal basolateral membrane's lipid.     

Formation of micelles and membrane action of the local anesthetic tetracaine hydrochloride

The formation of micelles of the local anesthetic tetracaine hydrochloride in aqueous phosphate buffer solution of pH 6.5 and ionic strength ($\text{I}$) 0.10 was examined at 22°C by surface tension and using the fluorescent indicators perylene (peri-dinaphthalene) and 8-anilino-1-naphthalene sulfonic acid, sodium salt (ANS). The critical micelle concentration was located at 0.069, 0.071 and 0.063 M by measurements of surface tension, perylene solubilization and enhancement of ANS fluorescence, respectively. In contrast to other cationic surfactants, the anesthetic monomer did not show evidence of forming a fluorescent molecular complex with ANS under the experimental conditions of this study.The formation of micelles by tetracaine-HCl showed a pronounced effect on lipid membranes by inducing an abrupt decrease in the scattered light of egg lecithin liposomes at an anesthetic concentration roughly similar to its critical micelle concentration. This optical behaviour is characteristic of liposome damage and can be interpreted to mean that the lipids become solubilized into tetracaine-HCl micelles.The ability of this local anesthetic to form micelles can be taken as a manifestation of the same hydrophobic forces that lead to partitioning of the drug into membranes.     

Nanosecond fluorescence anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene in membranes

Nanosecond decays of the fluorescence anisotropy, $\text{r}$, were studied for the emission of 1,6-diphenyl-l,3,5-hexatriene (DPH) embedded in a series of mixed multilamellar liposomes containing egg yolk phosphatidylcholine, phosphatidylethanolamine and cholesterol in varying molar ratios, as well as in membranes of intact cells and in virus envelopes.The relative contributions of the fast and the infinitely slow decaying component to the steady-state value, $\text{r}$, of the fluorescence anisotropy were very similar for artifical and biological membranes.Angles, θ, of the cone, by which the motion of the fluorescent molecule is limited, were calculated from the intensity of the infinitely slow decaying anisotropy component and compared with steady-state fluorescence anisotropies and with ‘microviscosities’, 〈η〉. An increase in 〈η〉 from 1.5 to 5.2 P in our systems was accompanied by a decrease in θ from 49° to 30° while the decrease in the mean motional relaxation times, $\text{φ}{}_{\mathrm{<mtext>f</mtext>}}$, of the label molecule was not more than 1 ns and due mainly to changes in the potential, by which the diffusion of DPH in the membrane is restricted. From these observations we conclude that differences in the steady-state fluorescence anisotropy and in ‘microviscosities’ of cholesterol-containing membranes ($\text{r}\text{> 0.15}$) represent changes in the degree of static orientational constraint rather than changes in diffusion rates of the label.     

Differential polarized phase fluorometric studies of phospholipid bilayers under high hydrostatic pressure

Differential polarized phase fluorometry was used to quantify the rotational rate ($\text{R}$) and limiting anisotropy ($\text{r}{}_{\mathrm{\infty }}$) of the membrane probe diphenylhexatriene (DPH) in solvents and lipid vesicles exposed to hydrostatic pressures ranging from 1 bar to 2 kbar. These measurements reveal the effect of pressure on the phase-transition temperatures of the phosphatidylcholine vesicles, and the effects of pressure on order parameter of the acyl side-chain region of the membranes, the latter as indicated by $\text{r}{}_{\mathrm{\infty }}$. In addition to the well-known elevation of the transition temperature ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) with pressure, our results demonstrate that increased pressure restores the order of the bilayers to that representative of temperatures below the transition temperature. We also found that solvents which allowed free isotropic rotation of DPH at 1 bar no longer allowed free rotation when sufficiently compressed; moreover, the apparent DPH rotational rate increased with $\text{r}{}_{\mathrm{\infty }}$. Pressure studies using both DPH and the charged DPH analogue, trimethylammonium DPH (TMA-DPH) indicated that the $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ of dipalmitoylphosphatidylcholine vesicles increased 23 K/kbar and an apparent volume change of 0.036 ml/mol lipid at the phase transition. Assuming, as has been proposed, that TMA-DPH is localized near the glycerol backbone region of the bilayers, these results indicate a similar temperature- and pressure-dependent phase transition in this region and the acyl side-chain region of the membrane.     

The quenching of an intramembrane fluorescent probe. A method to study the binding and permeation of phloretin through bilayers

Phloretin and phloretin-like dipolar non-electrolytes strongly quench the fluorescence of several membrane-bound probes, including 1,6-diphenylhexa-1,3,5-triene and anthroyl derivatives of long-chain fatty acids. Fluorescence intensity measurements therefore provide a simple and sensitive method to study the equilibrium binding properties and permeability of phloretin-like molecules in biological and artificial membrane systems. The dissociation constants for the binding of phloretin and naringenin to phosphatidylcholine vesicle membranes are determined, assuming the Stern-Volmer relation, from the fluorescence intensity of intramembrane probes as a function of phloretin and naringenin concentrations. Results (phloretin, $\text{9 ± 1}\text{μM}$; naringenin, $\text{21 ± 4}\text{μM}$) agree with the dissociation constants obtained using absorption titration performed in the absence of fluorescent probes. Fluorescence nanosecond lifetime measurements show that the mechanism of quenching of diphenylhexatriene and 16-anthroylpalmitic acid by phloretin and naringenin is largely diffusional in nature. The transmembrane movement of phloretin through phosphatidylcholine vesicles was observed by the stopped-flow technique, in which phloretin is mixed rapidly with a vesicle solution containing a membrane-bound fluorescent probe. The time course obtained by fluorescence measurements was identical to that obtained in a parallel measurement of the time course of optical absorption of phloretin. Stopped-flow data for the permeability of phosphatidylcholine liposomes and red blood cell membranes are also presented. The use of a membrane-bound indicator greatly extends the range of concentrations and pH values as well as the types of systems which can be characterized by optical means.     

Interaction of membrane surface charges with the reconstituted ADP/ATP-carrier from mitochondria

Various modulating influences of negative and positive membrane charges on binding and transport properties of the reconstituted ADP/ATP carrier from mitochondria were investigated. The results are interpreted in terms of functional and structural asymmetries of the adenine nucleotide carrier embedded in the liposomal membrane. The surface potential of liposomes was measured directly either by potential-dependent adsorption of the fluorescent dye $\text{2-p-}\text{toluidinylnaphthalene}$ 6-sulfonate (TNS) or by the $\text{p}\text{K}$ shift of the lipophilic pH indicator pentadecylumbelliferone. These results were correlated with the following observations. (1) Negative surface potentials increase the apparent dissociation constant, $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$, for binding of the negatively charged inhbitor carboxyatractylate to the reconstituted carrier protein. (2) Surface potentials modulate the apparent transport affinity, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, of the reconstituted adenine nucleotide carrier for ADP and ATP. The interaction of surface charges with the transport function was investigated with carrier proteins oriented both right-side-out and inside-out. Thus the influence of the surface potential on the function of the ADP/ATP carrier could be determined for the internal and external active sites of the translocator on the outer side of the membrane. Large discrepancies were observed not only between the potentials measured directly (fluorescent dyes) and those measured indirectly (binding and transport affinities), but also between the different surface potentials determined from the influence on the alternatively oriented carrier proteins. The effect of surface charges was rather weak on the cytosolic side of the translocator, whereas there was a strong influence of surface charges on the active site at the matrix side. The most obvious explanation, i.e., screening of negative membrane charges by positively charged amino acid residues at the protein surface, could be ruled out. Besides the modulation of binding affinities for substrates and inhibitors, an additional side-specific effect of surface charges on the transport velocity was observed. Again, the influence on the internal active site of the ADP/ATP carrier was found to be much higher than that on the cytosolic site. The observed effects can be explained by a definite structural asymmetry of the carrier embedded in the liposomal membrane. That site which is physiologically exposed to the cytosol is located at a considerable distance from the plane of the membrane, whereas the opposite site seems to be in close proximity to the membrane surface. Moreover, a spatial equivalence of carboxyatractylate binding site and nucleotide binding site at the external side of the carrier protein was concluded.     

Lectin and cholera toxin binding to guinea pig tumor (104C1) cell surfaces before and after glycosphingolipid incorporation

¹²⁵I-Labeled $\text{Dolichos biflorus}$ lectin and cholera toxin were used as probes for identification of Forssman- and GM1-type receptor sites on guinea pig tumor (104C1) cell surfaces. Increased binding of ¹²⁵I-labeled lectin and toxin to 104C1 cell surfaces was observed after the cells were treated with exogenous Forssman glycosphingolipid and GM1 ganglioside, respectively. Biosynthesis $\text{in vitro}$ of these two glycosphingolipids from their precursor molecules was established using a membrane preparation isolated from confluent cultures of guinea pig tumor 104C1 cells.     

The foundation of two distinct cell lineages within the mouse morula

The division of single cells, isolated from an 8-cell mouse embryo, to give $\text{2 ×}\text{1}\text{16}$ cells has been studied by sampling cells for analysis at defined stages during and after the division. Cells were analyzed for evidence of polarity in their surface organization as assessed by fluorescent ligand binding and distribution of microvilli. Individual $\text{1}\text{8}$ cells are polarized. At division, most (82%) divide such that both the pole of ligand binding and the pole of microvilli are distributed to only one of the two daughter cells. A couplet is thereby formed with a large polar cell and a small apolar cell. Some $\text{case1}\text{8}$ cells divide through the pole, generating a couplet of two polar cells, the poles being contiguous at the midbody. Elements of the surface polarity observed in the $\text{1}\text{8}$ cells can be found at all stages throughout division. Analysis of couplets of cells derived from newly formed 16-cell morulae also reveals that most consist of a polar:apolar pair and some consist of a polar:polar couplet in which the poles are contiguous at the midbody.The results indicate that two distinct cell populations are generated at division. These cells are known to occupy different positions within the morula, the polar cells being peripheral and the apolar cells being central. Since peripheral and central cells give rise to trophectoderm and inner cell mass in the blastocyst, we therefore suggest that the foundation of the trophectoderm and inner cell mass lineages may occur by a process of differential inheritance. This conclusion supports the recently proposed polarization hypothesis, which is discussed.     

Fluorescence spectroscopic studies of Huntington fibroblast membranes.

Using fluorescence spectroscopic methods, we compared the membrane properties of intact fibroblasts from both normal subjects and patients with Huntington disease (HD). Cells were stained with various fluorophores, including 1-anilino-8-naphthalene sulfonic acid (ANS), 2-toluidinyl-6-naphthalene sulfonic acid (TNS), 1,6-diphenyl-1,3,5-hexatriene (DPH), and 6-lauroyl-2-(dimethylamino)-naphthalene (LAURDAN). Using these labeled cells, we measured fluorescence yields and emission maxima (ANS, TNS, and LAURDAN), polarizations (TNS, DPH, and LAURDAN), lifetimes (TNS), and differential polarized lifetimes (DPH). In each instance, comparisons were made between cells from normal and from HD individuals. These cultures were controlled for passage number in culture and for age of donor. We found no significant differences between the HD and the control fibroblasts in experiments using the above-mentioned probes and spectroscopic parameters.     

Difference in microviscosity induced by different cholesterol levels in the surface membrane lipid layer of normal lymphocytes and malignant lymphoma cells

Microviscosity ($\text{}\text{?}$gh) in the surface membrane lipid layer of normal lymphocytes and malignant lymphoma cells, and in liposomes prepared from their lipid extracts, was determined with the aid of the fluorescence polarization properties of 1,6-diphenyl 1,3,5-hextriene embedded in it. The $\text{}\text{?}$gh values, both in intact cells and in the liposomes, are distinctively greater for normal lymphocytes than for the lymphoma cells, whereas the fusion activation energy in both types of cells and liposomes is 8 ± 0.5 kcal/mol. Determination of cholesterol revealed that its relative amount in a lymphoma cell is about half of that of a normal lymphocyte, a difference that may account for the above difference in fluidity. This thesis is supported by the observed changes in $\text{}\text{?}$gh, which follow artificial changes in cholesterol contents in the surface membrane of both cell types. Introduction of exogeneous cholesterol into the cell surface membranes was performed with lecithin-cholesterol (1:1) liposomes, and in lymphoma cells resulted in an increase of $\text{}\text{?}$gh to a level of normal lymphocytes. Extraction of native cholesterol from the cell surface membranes was carried out with lecithin liposomes, and in normal lymphocytes results in a decrease of $\text{}\text{?}$gh to a value similar to that of lymphoma cells. The induced changes in cholesterol contents are practically reversible for both cell types. By virtue of controlling the microviscosity of lipid layers, the level of cholesterol in cell surface membranes may play an important role in determining biological activities of normal and malignant cells.     

Fast kinetics of antagonist-acetylcholine receptor interactions: a temperature-jump relaxation study

The interactions of an antagonist with the membrane-bound acetylcholine receptor from $\text{Torpedo}$ have been studied using the temperature-jump relaxation method. The fluorescence emission of the antagonist, a pyrenyl-choline derivative, excited by energy transfer from the protein chromophores, enabled the observation of two relaxation processes in the lower millisecond time range. The concentration dependence of the relaxation times and associated amplitudes could be accounted for by a reaction scheme involving the binding of $\text{two}$ antagonist molecules and the subsequent isomerization of the complexes. Both binding steps appear to be diffusion-controlled and the second isomerization rate limiting. Competitive and non-competitive antagonism are discussed in relation to the proposed reaction mechanism.Recent advances in the study of the reaction mechanisms between the acetylcholine receptor (AChR)¹ and cholinergic ligands $\text{in}\text{vitro}$ have been accomplished by applying kinetic techniques (see review in (1)). In most cases extrinsic fluorescent probes have been employed in conjunction with the stopped-flow technique (2–7). Absorption spectroscopy has been used in one case (8). A slightly more direct approach utilized the intrinsic fluorescence of the membrane-bound AChR and cholinergic agonists of well characterized activity, including the natural neurotransmitter acetylcholine (9–11). From other potentially useful approaches or probes (see e.g. ref. (12)) only fragmentary and qualitative information is available. Since the introduction of dansyl-choline (13), and the subsequent demonstration of its mixed agonist-local anaesthetic action $\text{in}\text{vivo}$ (14), it became apparent that the pharmacological characterization of extrinsic fluorescent probes is an absolute prerequisite to the performance of any spectroscopic study $\text{in}\text{vitro}$. This prerequisite has been met for example in a later study (15) in which pyrene butyrylcholine was synthesized, having in mind the high molecular weight of the AChR oligomer and the need to match its rotational relaxation time with a chromophore of particularly long lifetime such as pyrene. This (15) and other (16) compounds of the series were found to be competitive $\text{antagonists}$ of the nicotinic AChR.In the present work one of these compounds is used to study the kinetics of the interaction with the membrane-bound AChR from $\text{Torpedo}\text{marmorata}$ by means of the temperature-jump relaxation technique. The simplest reaction mechanism accounting for the observed kinetics is discussed in relation to the available electrophysiological information on the action of this type of cholinergic blocking agent.     

The activation energy of incorporation of extrinsic probes in model vesicles

Time dependence of fluorescence enhancement of probes after addition to lipid vesicles has been used to investigate the position of chromophores in the lipid bilayer. Incorporation studies of a series of $\text{n-(9-}\text{anthroyloxy}$) fatty acids ($\text{n}\text{= 2, 2, 12}\text{and}\text{16}$) and 1,6-diphenylhexatriene in dipalmitoyl phosphatidylcholine vesicles are described. The activation energies for incorporation of these several lipid-mimic type fluorescent probes have been measured. Results show that the activation energy is a function of the distance of the anthracene moiety (chromophore) from the polar end of the probe and the length of the acyl portion of the probe. An average insertion energy of 0.6 kcal/carbon is seen for these fatty acid probes. The activation energy of 1,6-diphenylhexatriene, a factor of 2 greater than that of 16-(9-anthroyloxy)palmitic acid, is consistent with locating 1,6-diphenyl-hexatriene in the middle of the bilayer.     

Analysis of lectin-specific cell surface glycoprotein on neuroblastoma cells

The binding sites for the lectins wheat germ agglutinin, Ricinus communis agglutinin and concanavalin A on mouse neuroblastoma cell membranes were identified using SDS-gel electrophoresis in combination with fluorescent lectins. Ricinus communis agglutinin and wheat germ agglutinin were found to bind almost exclusively to a single polypeptide with an apparent molecular weight of 30 000. Concanavalin A labeled over 20 different polypeptides, most with molecular weights greater than 50 000. However, when the neuroblastoma cells were treated with concanavalin A so as to internalize all the concanavalin A binding sites visible at the level of the fluorescent microscope and the purified plasma membranes analyzed for their concanavalin A binding polypeptides, only four of the 20 glycopolypeptides were missing or significantly reduced in amount. Thus, these four high molecular weight concanavalin A-binding polypeptides appear to be the major cell surface receptors for concanavalin A. Binding studies with iodinated concanavalin A indicated that these polypeptides represented the high affinity concanavalin A binding sites $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 2 · 10}{}^{\mathrm{?7}}\text{M}$). Low affinity concanavalin A binding sites were present on the cell surface after internalization of high affinity concanavalin A binding sites.     

Fluorescent probe studies of mixed micelles of phospholipids and bile salts. Effect of cholesterol incorporation

The binding of the fluorescent alkylamines, $\text{N-(2-}\text{aminoethyl}\text{)-5-}\text{dimethylamino-1-naphthalene}$ sulfonamide, $\text{N-(5-}\text{aminopentyl}\text{)-5-}\text{dimethylamino-1-naphthalene}$ sulfonamide (dansyl cadaverine) and $\text{N-(10-}\text{aminodecyl}\text{)-5-}\text{dimethylamino-1-napthalene}$ sulfonamide with phospholipid and phospholipid-deoxycholate micelles, has been shown to increase with the length of the alkyl spacer chain. The probes bind more effectively to micelles containing unsaturated phospholipids and do not interact strongly with bile salt solutions at low concentrations. Cholesterol incorporation into mixed micelles results in a quenching of probe fluorescence due to displacement of probe molecules. The enhanced rigidity of the mixed micelles on solubilizing cholesterol is established by a decrease in pyrene excimer fluorescence and by the less effective perturbation of the micellar structure by 1-anilino-8-naphthalene sulfonate. The anionic probe 1-anilino-8-naphthalene sulfonate is also displaced from the mixed micelles when cholesterol is incorporated, suggesting a dominant role for packing and hydrophobic effects in binding both positively and negatively charged probes.     

Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes

The fluorescence quenching of the $\text{n-(9-}\text{anthroyloxy}\text{)}$ (AO) fatty acid probes has been investigated in aqueous dispersions, vesicles of egg phosphatidylcholine and vesicles formed from red cell ghosts. Negatively charged (KI), neutral (acrylamide) and positively charged (CuSO₄) quenchers were used to monitor the location of the probes. The fluorescence of the probes, with the exception of the shortest chain (11-(9-anthroyloxy)undecanoic acid) is not quenched by acrylamide when associated with vesicles. This indicates that in association with vesicles, the 9-anthroyloxy moiety of the long chain probes is buried within the hydrocarbon region and thus well shielded from the aqueous phase. Measurements with KI indicate that the probes are present in the membrane at depths corresponding to the position of the 9-anthroyloxy moiety on the fatty acid, and that the quencher itself forms a concentration gradient within the membrane. Very little or no CuSO₄ quenching was observed for $\text{n-}\text{(9-anthroyloxy)stearic}$ acid probes $\text{(n-}\text{AS)with}\text{n > 2}$, suggesting that in these vesicles Cu²⁺ does not significantly penetrate the bilayer.     

Cell surface antigens on erythroid cells: A comparison of normal and anemic (WW) mice

Cell surface antigens of normal and anemic ($\text{W}\text{W}$) mouse erythroid cells have been examined in cytotoxicity assays with two rat antisera. When tested on fetal liver cells, a rat anti-erythroblast serum recognized antigen(s) present on erythroid cells early in development, while rat anti-adult red blood cell serum recognized antigen(s) present on mature erythroid cells. Each of these sera had different activity on normal (+/+ or $\text{W}\text{+}$) as compared to anemic ($\text{W}\text{W}$) erythroid cells.