The work described here was funded by Fina Biosolutions LLC.

<ol>
	<li>Ellis, R. W., Granoff, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development and clinical uses of Haemophilus B conjugate vaccines. , (1994).</li><li>Goebel, W. F., Avery, O. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemo-immunological+studies+on+conjugated+carbohydrate-proteins.">Chemo-immunological studies on conjugated carbohydrate-proteins.</a> Journal of Experimental Medicine. 50 (4), 533-550 (1929).</li><li>Mond, J. J., Vos, Q., Lees, A., Snapper, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+independent+antigens.">T cell independent antigens.</a> Current Opinion in Immunology. 7 (3), 349-354 (1995).</li><li>Cruse, J. M., Lewis, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Conjugate Vaccines. 10, (1989).</li><li>Lees, A., Nelson, B. L., Mond, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+soluble+polysaccharides+with+1-cyano-4-dimethylaminopyridinium+tetrafluoroborate+for+use+in+protein-polysaccharide+conjugate+vaccines+and+immunological+reagents.">Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate for use in protein-polysaccharide conjugate vaccines and immunological reagents.</a> Vaccine. 14 (3), 190-198 (1996).</li><li>Shafer, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+soluble+polysaccharides+with+1-cyano-4-dimethylaminopyridinium+tetrafluoroborate+(CDAP)+for+use+in+protein-polysaccharide+conjugate+vaccines+and+immunological+reagents.+II.+Selective+crosslinking+of+proteins+to+CDAP-activated+polysaccharides.">Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for use in protein-polysaccharide conjugate vaccines and immunological reagents. II. Selective crosslinking of proteins to CDAP-activated polysaccharides.</a> Vaccine. 18 (13), 1273-1281 (2000).</li><li>Schneerson, R., Barrera, O., Sutton, A., Robbins, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization,+and+immunogenicity+of+Haemophilus+influenzae+type+b+polysaccharide-protein+conjugates.">Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates.</a> Journal of Experimental Medicine. 152 (2), 361-376 (1980).</li><li>Lees, A., Barr, J. F., Gebretnsae, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+soluble+polysaccharides+with+1-cyano-+4-dimethylaminopyridine+tetrafluoroborate+(CDAP)+for+use+in+protein-polysaccharide+conjugate+vaccines+and+immunological+reagents.+III+Optimization+of+CDAP+activation.">Activation of soluble polysaccharides with 1-cyano- 4-dimethylaminopyridine tetrafluoroborate (CDAP) for use in protein-polysaccharide conjugate vaccines and immunological reagents. III Optimization of CDAP activation.</a> Vaccines. 8 (4), 777 (2020).</li><li>Qi, X. -. Y., Keyhani, N. O., Lee, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+determination+of+hydrazine,+hydrazides,+and+their+mixtures+with+trinitrobenzenesulfonic+acid.">Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid.</a> Analytical biochemistry. 175 (1), 139-144 (1988).</li><li>Monsigny, M., Petit, C., Roche, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+determination+of+neutral+sugars+by+a+resorcinol+sulfuric+acid+micromethod.">Colorimetric determination of neutral sugars by a resorcinol sulfuric acid micromethod.</a> Analytical biochemistry. 175 (2), 525-530 (1988).</li><li>Hermanson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate Techniques. 3rd ed. , (2013).</li></ol>

Andrew Lees is founder and owner of Fina Biosolutions. He holds several patents relating to CDAP chemistry and benefits from licensing of the chemistry and CDAP conjugation know-how.

CDAP is a convenient reagent to derivatize and conjugate polysaccharides. This article describes the general method to use CDAP to derivatize polysaccharides with hydrazides (PS-ADH) and incorporates recently published improvements8. First, the technique emphasizes the importance of maintaining the target pH to control the activation process. We found that while many common buffers interfere with the CDAP activation reaction, DMAP could successfully be used as the buffer to manage the pH8. Furthermore, DMAP is already a reaction byproduct of CDAP activation. Finally, buffering the polysaccharide solution with DMAP before adding the CDAP facilitates precise targeting and maintenance of the reaction pH. As we describe, it is useful to adjust the pH of the concentrated DMAP stock solution such that when diluted, it reaches the targeted pH. Secondly, performing the process in the cold slowed the reaction time, making the activation process less frenetic and more forgiving. Lower temperature decreased the rate of CDAP hydrolysis, and the optimal activation time at pH 9 increases from ~3 min to ~15 min. In addition, less CDAP is required to achieve the same level of activation than when performed at room temperature. 
ADH-derivatized polysaccharides can be conjugated to proteins using carbodiimides (e.g., EDAC)7. For example, several licensed Haemophilus influenzae b (Hib) vaccines use the polyribosylribitolphosphate (PRP) derivatized with ADH to conjugate to tetanus toxoid using EDAC. CNBr was initially employed, but CDAP is a much easier reagent to use for this purpose. In our experience, a good target range for ADH derivatization is 10-30 hydrazides per 100 kDa polysaccharide or ~1-3% ADH by weight. 
The same process can be used to derivatize polysaccharides with primary amines by substituting the ADH for a diamine. It is recommended to use hexane diamine to derivatize polysaccharides with amines8. The aminated polysaccharide (PS-NH2) can be conjugated using reagents developed for protein conjugation11. Typically, the PS-NH2 is derivatized with a maleimide (e.g., succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) or N-&#947;-maleimidobutyryl-oxysuccinimide ester (GMBS)), and the protein is thiolated (e.g., with succinimidyl 3-(2-pyridyldithio)propionate (SPDP)). Thiol-maleimide chemistry is very efficient.
Proteins can also be directly coupled to CDAP-activated polysaccharides via the &#603;-amine on lysines. While the activation protocol used is generally similar to the one described here, it is necessary to optimize the level of activation, polysaccharide and protein concentration, as well as the protein:polysaccharide ratio5,6,8. 
Dextran is one of the easiest polysaccharides to activate with CDAP due to its relatively high density of hydroxyl groups, but some polysaccharides, such as Vi antigen, can be challenging. Consequently, there is no single "best" protocol for CDAP conjugation directly to proteins. We suggest first developing a protocol to achieve suitable levels of activation, as determined by the extent of hydrazide derivatization, and then proceeding to direct protein conjugation to CDAP-activated polysaccharide.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62597/activation-conjugation-soluble-polysaccharides-using-1-cyano-4">Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)</a>. A figure&#160;was updated.
Figure 4 was updated from:
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62597/62597fig04.jpg" /> 
Figure 4: Representative results for CDAP activation of dextran. Typical standard curves for the (A) resorcinol/sulfuric acid and (B) TNBS assays. The assay results for dextran activated with 0.25 and 0.5 mg CDAP/mg dextran are shown. Glucose was used as the standard for the resorcinol assay. Dextran, in mg/mL, is divided by 100 kDa to give a molar concentration. The hydrazide concentration is determined using ADH as the standard and the results expressed as &#181;M Hz.&#160;(C)&#160;Calculation of hydrazide: dextran ratios.The level of derivatization was calculated as hydrazides per 100 kDa of dextran to facilitate the comparison between polymers of different average molecular weights. The % weight ratio of g ADH/g dextran was calculated using a MW of 174 g/mole for ADH. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62597/62597fig4v4.jpg" /> 
Figure 4: Representative results for CDAP activation of dextran. Typical standard curves for the (A) resorcinol/sulfuric acid and (B) TNBS assays. The assay results for dextran activated with 0.25 and 0.5 mg CDAP/mg dextran are shown. Glucose was used as the standard for the resorcinol assay. Dextran, in mg/mL, is divided by 100 kDa to give a molar concentration. The hydrazide concentration is determined using ADH as the standard and the results expressed as &#181;M Hz.&#160;(C)&#160;Calculation of hydrazide: dextran ratios.The level of derivatization was calculated as hydrazides per 100 kDa of dextran to facilitate the comparison between polymers of different average molecular weights. The % weight ratio of g ADH/g dextran was calculated using a MW of 174 g/mole for ADH. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig4v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62597/activation-conjugation-soluble-polysaccharides-using-1-cyano-4">Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)</a>. A figure&#160;was updated.

Erratum: Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)

Conjugate vaccines, such as those consisting of polysaccharides covalently linked to a carrier protein, are among the remarkable advances in vaccinology1,2. Polysaccharides, as T-cell-independent antigens, are poorly immunogenic in infants and do not induce memory, class switching, or affinity maturation of antibodies3. These shortcomings are overcome in polysaccharide conjugate vaccines4. As most polysaccharides do not have a convenient chemical handle for conjugation, they must first be made reactive or &#34;activated.&#34; The activated polysaccharide is then linked either directly with the protein (or modified protein) or is functionalized for additional derivatization before conjugation4. Most licensed polysaccharide conjugate vaccines use either reductive amination or cyanylation to activate polysaccharide hydroxyls. Cyanogen bromide (CNBr), a reagent that had previously been used to activate chromatography resins, was initially used for polysaccharide derivatization. However, CNBr requires high pH, typically ~ pH 10.5 or greater, to partially deprotonate polysaccharide hydroxyls so that they are sufficiently nucleophilic to attack the cyano group. The high pH can be detrimental to base-labile polysaccharides, and neither CNBr nor the active cyano-ester initially formed is sufficiently stable at such high pH.
CDAP (1-cyano-4-dimethylaminopyridine tetrafluoroborate; Figure 1) was introduced by Lees et al. for use as a cyanylating agent for the activation of polysaccharides5,6. CDAP, which is crystalline and easy to handle, was found to activate polysaccharides at a lower pH than CNBr and with fewer side reactions. Unlike CNBr, CDAP-activated polysaccharides can be directly conjugated to proteins, simplifying the synthesis process. CDAP-activated polysaccharides can be functionalized with a diamine (e.g., hexane diamine) or a dihydrazide (e.g., adipic dihydrazide, ADH) to make amino- or hydrazide-derivatized polysaccharides. A high concentration of the homobifunctional reagent is used to suppress crosslinking of polysaccharides. Amino polysaccharides can then be conjugated using any of the myriad techniques used for protein conjugation. Hydrazide-derivatized polysaccharides are often coupled to proteins using a carbodiimide reagent (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC))7. Further optimization of CDAP polysaccharide activation has been described by Lees et al.8 and is incorporated into the protocol described here.
CDAP conjugation overview 
The CDAP protocol can be conceptualized as two phases: (1) the activation of the polysaccharide and (2) conjugation of the activated polysaccharide with a protein or ligand (Figure 2). The goal of the first step is to efficiently activate the polysaccharide, while the goal of the second is to efficiently conjugate to the activated polysaccharide. The activated polysaccharide ties the two steps together. This conceptualization helps focus on the critical elements of each step. Figure 2 expands on this conceptualization, showing the desired activation and coupling reactions, along with the hydrolysis reactions and side reactions.
During the activation phase, the three major concerns are CDAP stability, CDAP reaction with the polysaccharide hydroxyls, and the stability of the activated polysaccharide (Figure 3). CDAP hydrolysis increases with pH, as does the hydrolysis of the activated polysaccharide and the side reactions. However, the CDAP reaction with the polysaccharide is facilitated by increasing the pH. Efficiently activating polysaccharides with CDAP requires a balance between 1) the reactivity of the polysaccharide and CDAP and 2) the hydrolysis and side reactions of both the reagent and the activated polysaccharide.
In the original CDAP activation protocol described by Lees et al.5, CDAP activation of polysaccharides was carried out at room temperature in unbuffered pH 9 solution. The rate of activation was found to be rapid under this condition, and the activation would be complete within 3 min. The reaction was also accompanied by rapid hydrolysis of CDAP, causing a rapid pH drop of the unbuffered reaction solution. It was challenging to quickly raise and to maintain the reaction pH at the target value in such a short time frame. In the described protocol, activation was performed by adding CDAP from a 100 mg/mL stock solution to the unbuffered polysaccharide solution. The pH was raised 30 s later with &#34;an equal volume of 0.2 M triethylamine&#34;. Protein to be conjugated was then added after 2.5 min to the activation reaction. Notably, the pH of the activation step was not well controlled and most likely initially exceeded the target pH. The fast reaction requiring prompt pH adjustment made the activation process difficult to control and challenging to scale up.
In contrast to the original protocol, the modified protocol described here has two major improvements. First, the pH of the polysaccharide solution is pre-adjusted to target activation pH, using DMAP as the buffer, before the addition of CDAP. DMAP has a pKa of 9.5 and thus has good buffering power around pH 9, and unlike many other buffers, DMAP was not found to promote CDAP hydrolysis8. Furthermore, DMAP is already a process intermediate and therefore does not add a new component to the reaction mixture. Pre-adjusting the pH before adding CDAP eliminates the large pH swing at the beginning of the reaction and allows for more efficient maintenance of the target pH during the reaction. The second improvement is to perform the activation reaction at 0 &#176;C, where the rate of CDAP hydrolysis is markedly slower than that at room temperature. With the longer reagent half-life at 0 &#176;C, the activation time is increased from 3 min to 15 min to compensate for the slower activation rate at the lower temperature. The longer reaction time, in turn, makes it easier to maintain the reaction pH. The use of 0 &#176;C also slows the degradation of pH-sensitive polysaccharides, making it possible to prepare conjugates of this type of polysaccharide. The improvements in the protocol make the activation process less frenetic, easier to control, more reproducible, and more amenable to scaling up.
This article describes the improved protocol for carrying out controlled CDAP activation of polysaccharide at 0 &#176;C and at a specified target pH and performing subsequent derivatization of the activated polysaccharides with ADH. Also described is a trinitrobenzene sulfonic acid (TNBS) assay, based on the method of Qi et al.9, for the determination of hydrazide level on the modified polysaccharide. A modified assay for hexoses based on resorcinol and sulfuric acid10 is also described, which can be used for determining a broader range of polysaccharides. For more information on CDAP activation and conjugation, the reader is referred to earlier publications5,6,8 by Lees et al.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma</td>
			<td>34851</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipic acid dihydrazide</td>
			<td>Sigma</td>
			<td>A0638</td>
			<td>MW 174</td>
		</tr>
		<tr>
			<td>Amicon Ultra 15 10 kDa</td>
			<td>Millipore</td>
			<td>UFC901008</td>
			<td>MW cutoff can be 30 kDa for 200 kDa PS</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autotitrator or electronic pipet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker&#160; 2-4 L</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CDAP</td>
			<td>SAFC</td>
			<td>RES1458C</td>
			<td>Sigma</td>
		</tr>
		<tr>
			<td>DMAP</td>
			<td>Sigma</td>
			<td>107700</td>
			<td>MW 122.2</td>
		</tr>
		<tr>
			<td>Flake ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCl 1 M</td>
			<td>VWR</td>
			<td>BDH7202-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro stir bar</td>
			<td>VWR</td>
			<td>76001-878</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge tube (for CDAP)</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR</td>
			<td>BDH9286</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH 1 M</td>
			<td>Sigma</td>
			<td>1099130001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH 10 M</td>
			<td>Sigma</td>
			<td>SX0607N-6</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH probe</td>
			<td>Cole Parmer</td>
			<td>55510-22</td>
			<td>6 mm x 110 mm Epoxy single junction</td>
		</tr>
		<tr>
			<td>pH temperature probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipets &#38; tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Saline or PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small beaker 5-20 mL</td>
			<td>VWR</td>
			<td>10754-696</td>
			<td>A 10 mL beaker allows room for pH probe &#38; pipet</td>
		</tr>
		<tr>
			<td>Small ice bucket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stir plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Resorcinol assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Combitip</td>
			<td>Eppendorf</td>
			<td>10 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>DI water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing</td>
			<td>Repligen</td>
			<td>132650T</td>
			<td>Spectra/Por 6-8kDa</td>
		</tr>
		<tr>
			<td>Dialysis tubing clips</td>
			<td>Repligen</td>
			<td>142150</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Repeat pipettor</td>
			<td>Eppendorf</td>
			<td>M4</td>
			<td></td>
		</tr>
		<tr>
			<td>Resorcinol</td>
			<td>Sigma</td>
			<td>398047</td>
			<td></td>
		</tr>
		<tr>
			<td>Sugar standard</td>
			<td></td>
			<td>As appropriate</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Sulfuric acid 75%</td>
			<td>VWR</td>
			<td>BT126355-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TNBS assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adipic dihydrazide</td>
			<td>Sigma</td>
			<td>A0638</td>
			<td>MW 174</td>
		</tr>
		<tr>
			<td>Borosilcate test tubes 12 x 75</td>
			<td>VWR</td>
			<td>47729-570</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium borate, 0.5 M pH 9</td>
			<td>Boston Biologicals</td>
			<td>BB-160</td>
			<td></td>
		</tr>
		<tr>
			<td>TNBS 5% w/v</td>
			<td>Sigma</td>
			<td>P2297</td>
			<td>MW 293.17</td>
		</tr>
	</tbody>
</table>

activation and conjugation of soluble polysaccharides using 1-cyano-4-dimethylaminopyridine tetrafluoroborate (cdap)

Conjugate vaccines are remarkable advances in vaccinology. For the preparation of polysaccharide conjugate vaccines, the polysaccharides can be conveniently functionalized and linked to vaccine carrier proteins using 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), an easy-to-handle cyanylating reagent. CDAP activates polysaccharides by reacting with carbohydrate hydroxyl groups at pH 7-9. The stability and reactivity of CDAP are highly pH-dependent. The pH of the reaction also decreases during activation due to the hydrolysis of CDAP, which makes good pH control the key to reproducible activation. The original CDAP activation protocol was performed at room temperature in unbuffered pH 9 solutions.
Due to the rapid reaction under this condition (&#60;3 min) and the accompanying fast pH drop from the rapid CDAP hydrolysis, it was challenging to quickly adjust and maintain the target reaction pH in the short time frame. The improved protocol described here is performed at 0 &#176;C, which slows CDAP hydrolysis and extends the activation time from 3 min to ~15 min. Dimethylaminopyridine (DMAP) was also used as a buffer to pre-adjust the polysaccharide solution to the target activation pH before adding the CDAP reagent. The longer reaction time, coupled with the slower CDAP hydrolysis and the use of DMAP buffer, makes it easier to maintain the activation pH for the entire duration of the activation process. The improved protocol makes the activation process less frenetic, more reproducible, and more amenable to scaling up.

To illustrate the activation and derivatization of a polysaccharide using CDAP chemistry, dextran was activated at 0.25 and 0.5 mg CDAP/mg dextran. For each reaction, a 10 mg/mL dextran solution in water was chilled on ice, and 1/10th volume of a 2.5 M DMAP stock (prepared as described in section 3) was added. The final solution was brought to pH 9 by the addition of 0.1 M NaOH in 10 &#181;L aliquots. The solution was chilled and stirred, CDAP added, and the pH maintained at pH 9 by adding 10 &#181;L aliquots of 0.1 M NaOH for 15 min. Only 0.25 mL of 0.5 M ADH at pH 9 was added (less than the usual amount) and the reaction allowed to proceed overnight at 4 &#176;C.
The labeled dextran was then sequentially dialyzed against 1 M NaCl, 0.15 M NaCl, and water as described in section 6. The ADH-dextran was then assayed for dextran using the resorcinol/sulfuric acid assay (section 7.2). A typical standard curve using glucose as the sugar standard is shown in Figure 4A. The hydrazide content was determined using the TNBS assay described in section 7.3. A typical hydrazide standard curve using ADH as the standard is given in Figure 4B.
Representative calculations from the activation of dextran at the two levels of activation are shown in Figure 4A,B. The data are presented as both hydrazides per 100 kDa of dextran polymer and as a weight percent of ADH to dextran, as described in sections 7.9.3.4 and 7.9.3.5, respectively, in Figure 4C. The degree of derivatization approximately doubled as the CDAP ratio was doubled.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62597/62597fig01.jpg" /> 
Figure 1: Chemical structure of CDAP. CDAP = 1-cyano-4-dimethylaminopyridine tetrafluoroborate. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62597/62597fig02.jpg" /> 
Figure 2: Process of CDAP activation and conjugation. The process is conceptually divided into two phases, with the activated polysaccharide common to both. Under basic conditions, CDAP activates polysaccharide hydroxyls, releasing DMAP (reaction 1). CDAP hydrolysis also releases DMAP (reaction 3). Although a cyano-ester is shown, this may not be the actual intermediate. The intermediate is, therefore, referred to as (CDAP) &#34;activated&#34; polysaccharide. During the first activation phase, the activated polysaccharide can hydrolyze (reaction 4) or undergo side reactions (reaction 5). In the second conjugation phase (reaction 2), the activated polysaccharide reacts with an amine to form a stable isourea bond in addition to reactions 4 and 5. Abbreviations: CDAP = 1-cyano-4-dimethylaminopyridine tetrafluoroborate; DMAP = 4-dimethylaminopyridine; R-NH2 = amine. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62597/62597fig03.jpg" /> 
Figure 3: CDAP activation and conjugation. The process requires balancing CDAP reactivity with the polysaccharide, the stability of the CDAP and activated polysaccharide, as well as the reactivity of the activated polysaccharide with that of the amine. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62597/62597fig4v4.jpg" /> 
Figure 4: Representative results for CDAP activation of dextran. Typical standard curves for the (A) resorcinol/sulfuric acid and (B) TNBS assays. The assay results for dextran activated with 0.25 and 0.5 mg CDAP/mg dextran are shown. Glucose was used as the standard for the resorcinol assay. Dextran, in mg/mL, is divided by 100 kDa to give a molar concentration. The hydrazide concentration is determined using ADH as the standard and the results expressed as &#181;M Hz.&#160;(C)&#160;Calculation of hydrazide: dextran ratios.The level of derivatization was calculated as hydrazides per 100 kDa of dextran to facilitate the comparison between polymers of different average molecular weights. The % weight ratio of g ADH/g dextran was calculated using a MW of 174 g/mole for ADH. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig4v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate CDAP at JoVE.com

Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate CDAP

Proteins and amine-containing ligands can be covalently linked to polysaccharides activated by the cyanylation reagent, 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), to form covalent protein (ligand)-polysaccharide conjugates. This article describes an improved protocol for carrying out controlled CDAP activation at 0 &#176;C and varying pH and performing subsequent conjugation of the activated polysaccharides.

Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)

Conjugate vaccines are remarkable advances in vaccinology. For the preparation of polysaccharide conjugate vaccines, the polysaccharides can be ...

activation-conjugation-soluble-polysaccharides-using-1-cyano-4

Research

JoVE Journal

Immunology and Infection

6.2K Views.  Fina Biosolutions LLC. Proteins and amine-containing ligands can be covalently linked to polysaccharides activated by the cyanylation reagent, 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), to form covalent protein (ligand)-polysaccharide conjugates. This article describes an improved protocol for carrying out controlled CDAP activation at 0 °C and varying pH and performing subsequent conjugation of the activated polysaccharides. 

Video: Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate CDAP

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease severity in patients with autoimmune pulmonary alveolar proteinosis (PAP)1-3. As abrogation of GM-CSF bioactivity in the lung is the likely cause for autoimmune PAP4,5, it is promising to measure the neutralizing capacity of GM-CSF autoantibodies for evaluating the disease severity in each patient with PAP.
Until now, neutralizing capacity of GM-CSF autoantibodies has been assessed by evaluating the growth inhibition of human bone marrow cells or TF-1 cells stimulated with GM-CSF6-8. In the bioassay system, however, it is often problematic to obtain reliable data as well as to compare the data from different laboratories, due to the technical difficulties in maintaining the cells in a constant condition.
OBJECTIVE: To mimic GM-CSF binding to GM-CSF receptor on the cell surface using cell-free receptor-binding-assay.
METHODS: Transgenic silkworm technology was applied for obtaining a large amount for recombinant soluble GM-CSF receptor alpha (sGMR&alpha;) with high purity9-13. The recombinant sGMR&alpha; was contained in the hydrophilic sericin layers of silk threads without being fused to the silk proteins, and thus, we can easily extract from the cocoons in good purity with neutral aqueous solutions14,15. Fortunately, the oligosaccharide structures, which are critical for binding with GM-CSF, are more similar to the structures of human sGMR&alpha; than those produced by other insects or yeasts.
RESULTS: The cell-free assay system using sGMR&alpha; yielded the data with high plasticity and reliability. GM-CSF binding to sGMR&alpha; was dose-dependently inhibited by polyclonal GM-CSF autoantibody in a similar manner to the bioassay using TF-1 cells, indicating that our new cell-free assay system using sGMR&alpha; is more useful for the measurement of neutralizing activity of GM-CSF autoantibodies than the bioassay system using TF-1 cell or human bone marrow cells.
CONCLUSIONS: We established a cell-free assay quantifying the neutralizing capacity of GM-CSF autoantibody.

We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.