Name: myeloid innate signaling pathway regulation by malt1 paracaspase activity
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 7 min 9 s
Description: Watch this Scientific Journal Video about Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity at JoVE.com

We thank Elsevier for their authorization (license number 4334770630127) to reproduce here Figure 2A from Unterreiner et al. (2017).

Experiments were conducted according to the guidelines and standards of the Novartis Human Research Ethics Committee.
1. Preparation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Buffy Coats
NOTE: We received buffy coats from healthy volunteers one day after collection, in 50 mL bags. They were provided under informed consent and collected through the Interregionale Blutspende Schweizeriches Rotes Kreuz. We handled them using the procedure below, at room temperature unless specified otherwise.
<ol>
	<li>Prepare a sterile and clean pair of scissors and a 1 L beaker with a plastic bag (under a lamina flow).</li>
	<li>Transfer the buffy coat into the beaker and carefully open it with the scissors.</li>
	<li>Using a 25 mL pipette, add 100 mL of phosphate-buffered saline buffer/ethylenediaminetetraacetic acid (PBS/EDTA: PBS 1x pH 7.4 containing no CaCl2 and no MgCl2, supplemented with 2 mM EDTA pH 8.0). 
	NOTE: Using the same pipette and after slowly pipetting the solution up and down, dispense 25 mL of the diluted buffy coat into 6 conical centrifuge tubes of 50 mL pre-filled with 15 mL of polysaccharide-based density gradient.</li>
	<li>Centrifuge 20 min at 800 x g with moderate acceleration (set at 4 out of 9) and without brake to allow for separation of cells based on their densities. 
	NOTE: After centrifugation, three layers will be visible; a pellet containing red blood cells and granulocytes, an upper layer made of plasma, and in between a white ring containing peripheral blood mononuclear cells (PBMCs).</li>
	<li>Harvest the PBMC ring using a 10 mL pipette and transfer to new 50 mL tubes. 
	NOTE: Two to three 50 mL tubes are normally required per buffy coat. At this stage, some plasma likely contaminates the collected PBMCs, which should have no impact on the subsequent enrichment steps.</li>
	<li>Top up to 50 mL every tube using PBS/EDTA and proceed to three successive washes with decreasing centrifugation time and speed (15 min at 520 x g, 10 min at 330 x g, 8 min at 150 x g). 
	NOTE: At each step, the supernatant is poured off in a liquid waste container and the cell pellet is resuspended in 50 mL of PBS/EDTA (pellets may be pooled after the first wash).</li>
	<li>After the final wash, resuspend the pellet in 25 mL of an ice-cold lysis buffer (see Table of Materials) to lyse the red blood cells by osmotic pressure.</li>
	<li>Incubate until the solution becomes clear (&#8804;5 min at room temperature).</li>
	<li>Stop the reaction by adding 25 mL of separation buffer (PBS 1x pH 7.4 containing no CaCl2 and no MgCl2, supplemented with 2% heat-inactivated fetal bovine serum (FBS) and 1 mM EDTA pH 8.0).</li>
	<li>Wash one more time at 150 x g for 8 min. 
	NOTE: At this stage PBMCs can be used as a bulk population (see step 3: PBMCs and monocytes treatments and stimulatory conditions) or processed for monocyte enrichment (see step 2: Preparation of monocytes from PBMCs), or frozen down for subsequent use (see step 5: Monocytes and PBMCs freezing/thawing procedures).</li>
</ol>
2. Preparation of Monocytes from PBMCs
<ol>
	<li>Resuspend the PBMCs obtained at step 1.10 in separation buffer and count them to reach 5 x 107cells/mL.</li>
	<li>Transfer the cell suspension into a 14 mL conical centrifuge tube with cap.</li>
	<li>Add 50 &#181;L of monocyte enrichment antibody cocktail per mL of cell suspension, vortex, and incubate 10 min at 4 &#176;C.</li>
	<li>Add 50 &#181;L of monocyte enrichment beads per mL of cells. 
	NOTE: The beads must be thoroughly vortexed to ensure homogeneity of the suspension.</li>
	<li>After adding the beads, shortly vortex the cell suspension and incubate 5 min at 4 &#176;C.</li>
	<li>Rinse the upper part of the tube with separation buffer until the tube is filled up to 10 mL.</li>
	<li>Slowly mix the solution by pipetting up and down.</li>
	<li>Place the tube without cap into a separation magnet.</li>
	<li>Incubate for 2.5 min at room temperature.</li>
	<li>Pour slowly into a 15 mL conical centrifuge tube.</li>
	<li>At this stage, use monocytes directly (see step 3: PBMCs and monocytes treatments and stimulatory conditions) or differentiate them into immature Monocyte-derived Dendritic Cells (iMoDCs) (see step 4), or freeze them down for subsequent use (see step 5).</li>
</ol>
3. PBMCs and Monocytes Treatments and Stimulatory Conditions
<ol>
	<li>Count cells and dilute them in culture medium (Roswell Park Memorial Institute medium (RPMI) 10% FBS + 1 mM Sodium Pyruvate + 100 U/mL Penicillin Streptomycin (Pen/Strep) + 5 &#181;M &#946;-mercaptoethanol), down to 1.25 x 104 cells/well.</li>
	<li>Distribute 30 &#181;L of cell suspension per well of a 384-well plate.</li>
	<li>Add 15 &#181;L of 4x concentrated compound solutions and pre-incubate for 1 h at 37 &#176;C, 5% CO2.</li>
	<li>Add lipopolysaccharide (LPS) to a final concentration of 10 ng/mL, or depleted zymosan (DZ) to a final concentration of 100 &#181;g/mL, or keep in plain medium.</li>
	<li>Incubate overnight at 37 &#176;C, 5% CO2.</li>
	<li>Take 10 &#181;L of the supernatant to measure secreted TNF-a levels (see step 7: Cytokines and viability measurements).</li>
</ol>
4. Monocytes Differentiation into iMoDCs and Stimulatory Conditions
<ol>
	<li>Count monocytes (enriched at step 2) and centrifuge the cell suspension at 520 x g for 5 min.</li>
	<li>Pipette off the supernatant and add culture medium (RPMI+ 10% FBS to obtain a final cell suspension of 0.4 x 106 cells/mL.</li>
	<li>Add 80 ng/mL recombinant human IL-4 + 100 ng/mL GM-CSF.</li>
	<li>Dispense 5 mL of cell suspension per well in a 6-well plate.</li>
	<li>Incubate for 7 days at 37 &#176;C, 5% CO2.</li>
	<li>At day 7, harvest cells by pipetting gently to avoid their activation.</li>
	<li>Centrifuge at 520 x g for 5 min.</li>
	<li>Vacuum aspirate and resuspend in 50 mL of culture medium without growth factors.</li>
	<li>Centrifuge at 520 x g for 5 min and resuspend at 1 x 106 cells/mL in culture medium.</li>
	<li>Dispense 100 &#181;L of cell suspension (105 cells) per well of a 96-well flat bottom plate.</li>
	<li>Pre-incubate for 1 h at 37 &#176;C after adding 50 &#181;L of 4x concentrated compound solutions, prepared as described at step 8: Compounds preparation.</li>
	<li>Add 50 &#181;L of stimuli (4x concentrated), prepared as described at step 9.</li>
	<li>Incubate 24 h at 37 &#176;C.</li>
	<li>Mix each well and transfer cells and supernatant (SN) into a 96-well V-bottom plate.</li>
	<li>Spin down at 475 x g for 5 min.</li>
	<li>Transfer supernatant into a new 96-well flat bottom plate, seal and freeze at -20 &#176;C until further use.</li>
</ol>
5. Monocytes and PBMCs Freezing/Thawing Procedures
<ol>
	<li>Freezing
	<ol>
		<li>Spin down PBMC or monocyte cell preparations at 520 x g for 5 min.</li>
		<li>Vacuum aspirate the supernatant and resuspend cells in freezing medium at 1 x 107 cells/mL.</li>
		<li>Dispense 1 mL of cell suspension into cryotubes, transfer the tubes to a specific cooling device (see Materials table) and place it at -80&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Thawing
	<ol>
		<li>Thaw the cryotube and quickly transfer its content into a 15 mL conical centrifuge tube containing 9 mL of culture medium.</li>
		<li>Centrifuge at 520 x g for 5 min.</li>
		<li>Vacuum aspirate supernatant and resuspend cell pellet in 5 mL of culture medium. Cells are now ready for further experimental processing.</li>
	</ol>
	</li>
</ol>
6. Mouse Spleen Cells Preparation and Treatment
NOTE: We conducted animal sacrifices according to the guidelines and standards of the Novartis Animal Welfare Organization. Studies were approved by the Ethics Committee of the regional governmental authority (Kantonales Veterin&#228;ramt der Stadt Basel). We sacrificed animals by isoflurane over-exposure, with all efforts made to minimize suffering.
<ol>
	<li>Harvest the spleen and dissociate tissue using a tube equipped with a mechanical tissue grinder device and filled with 5 mL of cold RPMI medium.</li>
	<li>Use the spleen program of the dissociation machine to grind organs.</li>
	<li>Filter the cells through a 100 mm nylon cell strainer.</li>
	<li>Transfer the suspension into 50 mL tubes and centrifuge at 4 &#176;C for 10 min at 320 x g.</li>
	<li>Vacuum aspirate the supernatant, resuspend the cell pellet in 3 mL of ice-cold lysis buffer, and incubate for &#8804;2 min on ice.</li>
	<li>Stop lysis by adding 7 mL of RPMI medium.</li>
	<li>Filter again through a 100 &#181;m nylon cell strainer.</li>
	<li>Spin down cell suspension at 330 x g for 10 min at 4 &#176;C.</li>
	<li>Vacuum aspirate supernatant and resuspend cells at 11 x 106 cells/mL in complete medium (RPMI supplemented with 10% FBS, 100 U/mL Pen/Step and 5 &#181;M &#946;-Mercaptoethanol).</li>
	<li>Plate 1 x 106 cells/well (90 &#181;L) in a 96-well plate (flat bottom).</li>
	<li>Add 5 &#181;L of 20x concentrated compound solution previously diluted in RPMI medium as described in step 8.3: Serial dilution for murine spleen cells.</li>
	<li>Incubate for 30 min at 37 &#176;C, 5% CO2.</li>
	<li>Add 5 &#181;L of 20x concentrated DZ (final concentration 30 &#181;g/mL) or 20x concentrated LPS + IFN-g (TLR-4) (final concentration 1 &#181;M LPS and 10 ng/mL IFN-g).</li>
	<li>Incubate overnight at 37 &#176;C, 5% CO2.</li>
	<li>Centrifuge at 330 x g for 10 min.</li>
	<li>Transfer supernatants in new plates, seal and freeze at -20 &#176;C until further use.</li>
</ol>
7. Cytokines and Viability Measurements
<ol>
	<li>Human TNF-a measurement by HTRF (Homogenous Time Resolved Fluorescence) 
	NOTE: The protocol followed the supplier&#8217;s recommendations, briefly summarized below.
	<ol>
		<li>Mix 1 volume of reconstituted reagent (anti TNF-a-cryptate and anti-TNF-a-XL665) with 19 volumes of reconstitution buffer (50 mM phosphate buffer pH 7.0, 0.8 M potassium fluoride (KF), 0.2% bovine serum albumin (BSA)).</li>
		<li>Mix the two ready-to-use antibody solutions 1:1 just prior to dispensing the reagents.</li>
		<li>Dispense 10 &#181;L of supernatants from step 3.6 into white 384-well plates.</li>
		<li>Dispense 10 &#181;L of the antibody mix.</li>
		<li>Cover the plate with a sealer and incubate at 4 &#176;C overnight.</li>
		<li>Read plate on a microplate reader (50&#8211;200 flash).</li>
	</ol>
	</li>
	<li>Human IL-23 measurement by HTRF (Homogenous Time Resolved Fluorescence) 
	NOTE:&#160;The protocol followed the supplier&#8217;s recommendations, briefly summarized below.
	<ol>
		<li>Mix one volume of reconstituted reagent (anti IL-23-cryptate-antibody and anti-IL-23 D2-antibody) with 19 volumes of detection buffer #3.</li>
		<li>Mix the two ready-to-use antibody solutions 1:1 just prior to dispensing the reagents.</li>
		<li>Dispense 10 &#181;L of supernatants from step 3.6 into white 384-well plates.</li>
		<li>Dispense 10 &#181;L of the antibody mix.</li>
		<li>Cover the plate with a sealer and incubate at 4 &#176;C overnight.</li>
		<li>Read plate on a microplate reader (50&#8211;200 nm flash).</li>
	</ol>
	</li>
	<li>Human IL-6, IL-8, IL-1&#946; and TNF-&#945; measurements by electrochemiluminescence 
	NOTE: All samples were diluted at 1/150 in diluent 2 (first dilution: 10 &#181;L in 150 &#181;L, then 20 &#181;L in 180 &#181;L). The protocol followed the supplier&#8217;s recommendations:
	<ol>
		<li>Dilute samples and standard in diluent 2.</li>
		<li>Proceed to dilution of the standard in diluent 2 using a 1/4 serial fold dilution.</li>
		<li>Wash plates three times with wash buffer.</li>
		<li>Dispense 50 &#181;L of samples or standard per well.</li>
		<li>Incubate for 2 h at room temperature under agitation.</li>
		<li>Wash the plate four times with PBS + 0.05% Polysorbate 20.</li>
		<li>Add 25 &#181;L of detection antibody (60 &#181;L of each antibody for 3 mL final) in diluent 3.</li>
		<li>Incubate for 2 h at room temperature under agitation.</li>
		<li>Wash the plate four times with PBS + 0.05% Polysorbate 20.</li>
		<li>Avoiding bubbles, add 150 &#181;L per well of read buffer (Tris-based buffer containing tripropylamine, diluted 2x in ddH20) as a co-reactant for light generation in electrochemiluminescence immunoassays.</li>
		<li>Read the plate (without delay) on a multiplex plate reader.</li>
	</ol>
	</li>
	<li>Mouse TNF-a measurement by ELISA following the supplier&#8217;s protocol
	<ol>
		<li>Dilute supernatant 1:1 in assay diluent (ready to use protein-containing buffer).
		<ol>
			<li>Prepare reagents, samples, and standard dilutions as described in the kit. Add 50 &#956;L of assay diluent to each well.</li>
			<li>Add 50 &#956;L of standard, control, or sample per well.</li>
		</ol>
		</li>
		<li>Mix by gently tapping the plate frame for 1 min.</li>
		<li>Cover with the provided adhesive strip and incubate for 2 h at room temperature.</li>
		<li>Aspirate each well and wash with 400 &#956;L/well (repeat this step five times in total).</li>
		<li>After the final wash, remove any remaining wash buffer by aspirating.</li>
		<li>Invert the plate and blot against clean paper towels.</li>
		<li>Add 100 &#956;L of mouse TNF-&#945; conjugate to each well. Cover with a new adhesive strip.</li>
		<li>Incubate for 2 h at room temperature.</li>
		<li>Repeat the aspiration/wash as in step 7.4.4.</li>
		<li>Add 100 &#956;L of substrate solution to each well and incubate for 30 min at room temperature protected from light.</li>
		<li>Add 100 &#956;L of diluted hydrochloric acid solution (stop solution) to each well. Gently tap the plate to ensure thorough mixing.</li>
		<li>Measure the optical density at 450 nm (with the correction wavelength set at 560 nm) using a microplate reader (to be done within 30 min).</li>
	</ol>
	</li>
	<li>Cell viability
	<ol>
		<li>After removing supernatants from PBMC or monocyte preparations, assess cell viability using a ready to use resazurin solution (oxidation-reduction indicator) added directly to the cell suspension to 10% final concentration.</li>
		<li>Incubate for 1 to 2 h at 37 &#176;C, 5% CO2.</li>
		<li>Read fluorescence at 590 nm (Excitation 540 nm) using a microplate reader.</li>
	</ol>
	</li>
</ol>
8. Compound Preparation
<ol>
	<li>Serial dilution for iMoDCs
	<ol>
		<li>Dilute the MLT-827 stock solution (10 mM in dimethyl sulfoxide (DMSO) into medium to reach 8 &#181;M in one go (4x concentrated).</li>
		<li>Perform a six-step 1:5 serial dilution, using medium + 0.08% DMSO. Use the same medium + 0.08% DMSO solution for the vehicle (no compound) condition.</li>
	</ol>
	</li>
	<li>Single dose testing for iMoDCs
	<ol>
		<li>Dilute the MLT-827, AFN700 and Cpd11 stock solutions into medium to reach 4 &#181;M in one go (4x concentrated).</li>
	</ol>
	</li>
	<li>Serial dilution for murine spleen cells
	<ol>
		<li>Dilute a 10 &#181;M MLT-827 solution (obtained from a 10 mM stock solution following a one go dilution in medium) to 0.01 &#181;M, with a DMSO end concentration of 0.1%.</li>
		<li>For the dilution steps, take 2 &#181;L of each dilution, add 38 &#181;L RPMI, and pipet 5 &#181;L into the well. 
		NOTE: All treatments are performed in triplicate.</li>
	</ol>
	</li>
</ol>
9. Stimuli Preparation
<ol>
	<li>Depleted zymosan (DZ)
	<ol>
		<li>Add 2 mL of sterile endotoxin-free water to 10 mg of DZ.</li>
		<li>Vortex to homogenize the stock solution, vortex also prior each use.</li>
		<li>Aliquot solution and store aliquots at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Trehalose-6,6-dibehenate (TDB)
	<ol>
		<li>Add 100 &#181;L of DMSO to 1 mg TDB, heat at 60 &#176;C in a water bath for 15&#8211;30 s.</li>
		<li>Vortex and immediately add 900 &#181;L of sterile PBS, vortex again.</li>
		<li>Heat for 10&#8211;15 min at 60 &#176;C and homogenize by vortexing prior each use.</li>
		<li>Keep the solution at 4 &#176;C.</li>
		<li>Perform serial dilutions and single dose preparation as for the MLT-827 compound. 
		NOTE: Because TDB needs to be prepared in DMSO, a final 1% DMSO concentration is present during cell stimulation.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In this work, we used simple experimental settings to study signaling pathways in human and mouse innate cells, and interrogate their dependency on MALT1 proteolytic function. Expanding on previous work11, our study showed that MALT1 paracaspase activity controls C-type lectin-like receptor induced cytokine production, including TNF-&#945;. In contrast, TLR-4-induced TNF-&#945; was independent of MALT1 in both species. Collectively, these data corroborated the key and selective contribution of the...

The paracaspase activity of MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) was revealed in 20081,2. Since then, a number of studies have reported its critical contribution to antigen receptor responses in lymphocytes. Genetic models in the mouse as well as pharmacology data support a key role in T cells, in T-cell dependent autoimmunity and in B-cell lymphoma settings3,4. In lymphocytes, MALT1 paracaspase activation occurs upon assembly of a CARD11-BCL10-MALT1 complex5, which is triggered by antigen-receptor proximal signaling downstream of the T- or B- cell receptor. There is also ample evidence that a similar CARD9-BCL10-MALT1 complex is important for propagating signals downstream of C-type lectin-like receptors (CLLR), e.g., Dectin-1, Dectin-2 and MINCLE in myeloid cells6,7. Dectin-1 has been particularly well studied because this pathway is critical for host defense against fungal infections8,9. Implication of MALT1 in Toll-like receptor (TLR) pathways, however, has remained controversial10. Recent evidence in human myeloid cells ruled out a direct role for MALT1 paracaspase activity in the regulation of TNF- production downstream of TLR-411.
In the present work, we used various experimental settings and stimulatory conditions in human and mouse myeloid cells to probe innate signaling pathways, relying on specific pharmacological tool inhibitors and measurement of cytokine production.

<table><tbody><tr><td>100 &amp;micro;m nylon cell strainer</td><td>Sigma</td><td>CLS431752</td><td></td></tr><tr><td>14 mL Falcon tube</td><td>BD Falcon</td><td>352057</td><td></td></tr><tr><td>15 mL Falcon tube</td><td>Falcon</td><td>352090</td><td></td></tr><tr><td>50 mL Falcon tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>6 well plates</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>96 well flat-bottom plate, with low evaporation lid</td><td>Costar</td><td>3595</td><td></td></tr><tr><td>96 well V-bottom plate</td><td>Costar</td><td>734-1798</td><td></td></tr><tr><td>Ammonium Chloride - NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma</td><td>A9434</td><td></td></tr><tr><td>Assay diluent RD1-W ELISA</td><td>R&amp;amp;D</td><td>895038</td><td>Assay diluent</td></tr><tr><td>Cell culture microplate, 384 well, black</td><td>Greiner</td><td>781986</td><td></td></tr><tr><td>Depleted Zymosan</td><td>Invivogen</td><td>tlrl-dzn</td><td>now: tlrl-zyd</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td>DMSO</td></tr><tr><td>EDTA-Na&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>E5134</td><td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td></tr><tr><td>ELISA muTNF-&amp;alpha;</td><td>R&amp;amp;D</td><td>SMTA00</td><td></td></tr><tr><td>Ficoll-Paque Plus</td><td>GE Healthcare</td><td>17-1440-03</td><td></td></tr><tr><td>gentleMACS C tubes</td><td>MACS Miltenyi Biotec</td><td>130-096-334</td><td></td></tr><tr><td>gentleMACS dissociator</td><td>MACS Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>GM-CSF</td><td>Novartis</td><td>-</td><td></td></tr><tr><td>Heat-inactivated Fetal bovine serum</td><td>Gibco</td><td>10082</td><td>FBS</td></tr><tr><td>HTRF hu IL-23</td><td>CisBio</td><td>62HIL23PEG</td><td></td></tr><tr><td>HTRF hu TNF-&amp;alpha;</td><td>CisBio</td><td>62TNFPEC</td><td></td></tr><tr><td>HTRF reconstitution buffer</td><td>CisBio</td><td>62RB3RDE</td><td>50mM Phosphate buffer, pH 7.0, 0.8M KF, 0.2% BSA</td></tr><tr><td>IFN-&amp;gamma;</td><td>R&amp;amp;D</td><td>L4516</td><td></td></tr><tr><td>IL-4</td><td>Novartis</td><td>-</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott</td><td>Forene</td><td></td></tr><tr><td>Lipopolysaccharides (LPS)</td><td>Sigma</td><td>L4391</td><td>LPS used in human samples</td></tr><tr><td>Lipopolysaccharides</td><td>Sigma</td><td>L4516</td><td>LPS used in murine samples</td></tr><tr><td>Lysis buffer</td><td>Self-made</td><td>-</td><td>155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA, pH 7.4</td></tr><tr><td>Magnet</td><td>Stemcell</td><td>18001</td><td></td></tr><tr><td>Microplate, 384 well white</td><td>Greiner</td><td>784075</td><td></td></tr><tr><td>Monocytes enrichment kit</td><td>Stemcell</td><td>19059</td><td></td></tr><tr><td>Nalgene Mr. Frosty Cryo 1&amp;deg;C Freezing Container</td><td>Nalgene</td><td>5100-0001</td><td>cooling device (containing Propanol-2)</td></tr><tr><td>PBS 1x pH 7.4 [-] CaCl&lt;sub&gt;2&lt;/sub&gt; [-] MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Gibco</td><td>10010</td><td>Phosphate-buffered saline</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td>Pen/Strep</td></tr><tr><td>Potassium bicarbonate - KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>P9144</td><td></td></tr><tr><td>PrestoBlue</td><td>Invitrogen</td><td>A13262</td><td>Resazurin solution for viability assessment</td></tr><tr><td>Propanol-2</td><td>Merck</td><td>1.09634</td><td></td></tr><tr><td>Read buffer</td><td>MesoScale Discovery</td><td>R92TC-3</td><td>Tris-based buffer containing tripropylamine</td></tr><tr><td>Recovery cell culture freezing medium</td><td>Gibco</td><td>12648-010</td><td>freezing medium</td></tr><tr><td>Roswell Park Memorial Institute Medium (RPMI) with Glutamax</td><td>Gibco</td><td>61870</td><td>+ 10% FBS for iMoDCs&lt;br/&gt; + 10% FBS + 1 mM Sodium Pyruvate + 100 U/mL Pen/Strep + 5 &amp;micro;M &amp;beta;-mercaptoethanol for human PBMCs and monocytes&lt;br/&gt; + 10% FBS + Pen/Strep + 5 &amp;micro;M &amp;beta;-mercaptoethanol for murine splenocytes</td></tr><tr><td>Separation buffer</td><td>Self-made</td><td>-</td><td>PBS pH 7.4 + 2% FBS + 1 mM EDTA pH 8.0</td></tr><tr><td>Sodium Pyruvate</td><td>Gibco</td><td>11360</td><td></td></tr><tr><td>Trehalose-6,6-dibehenate</td><td>Invivogen</td><td>tlrl-tdb</td><td>TDB</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P7949</td><td>Polysorbate 20</td></tr><tr><td>UltraPure 0.5 M EDTA pH 8.0</td><td>Invitrogen</td><td>15675</td><td>Ethylenediaminetetraacetic acid</td></tr><tr><td>Viewseal sealer</td><td>Greiner BioOne</td><td>676070</td><td></td></tr><tr><td>V-PLEX Proinflammatory Panel 1 Human Kit</td><td>MesoScale Discovery</td><td>K15049D</td><td>electrochemiluminescent multiplex assay (IL-1&amp;beta;, TNF-&amp;alpha;, IL-6, IL-8)</td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Gibco</td><td>31350</td><td></td></tr></tbody></table>

myeloid innate signaling pathway regulation by malt1 paracaspase activity

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-&#954;B activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-&#954;B activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor &#8212;signaling by contrast to TLR-signaling.

In myeloid cells, MALT1 relays activation signals downstream of several C-type lectin-like receptors, such as Dectin-1, Dectin-2 and MINCLE6. These pathways rely on (hem)ITAM motif-containing receptors (e.g., Dectin-1) or ITAM motif-containing co-receptors (e.g., FcR&#947;, for Dectin-2 and MINCLE) that recruit and activate the SYK kinase (Figure 1). This leads to activation of a protein kinase C isoform, namely PKC&#...

Watch this Scientific Journal Video about Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity at JoVE.com

Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells ...

myeloid-innate-signaling-pathway-regulation-malt1-paracaspase

Research

JoVE Journal

Biology

7.4K Views.  Novartis Institutes for BioMedical Research. MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.

Video: Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

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 ...

Immunology and Infection

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

Biochemistry

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Neuroscience

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Developmental Biology

Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple myeloma, induce ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase for proteasomal degradation. IMiDs have recently been utilized for the generation of bifunctional proteolysis targeting chimeras (PROTACs) to target other proteins for ubiquitination and proteasomal degradation by the CRBN E3 ligase. We designed and synthesized pomalidomide-based homobifunctional PROTACs and analyzed their ability to induce self-directed ubiquitination and degradation of CRBN. Here, CRBN serves as both, the E3 ubiquitin ligase and the target at the same time. The homo-PROTAC compound 8 degrades CRBN with a high potency with only minimal remaining effects on IKZF1 and IKZF3. CRBN inactivation by compound 8 had no effect on cell viability and proliferation of different multiple myeloma cell lines. This homo-PROTAC abrogates the effects of IMiDs in multiple myeloma cells. Therefore, our homodimeric pomalidomide-based compounds may help to identify CRBN&#8216;s endogenous substrates and physiological functions and investigate the molecular mechanism of IMiDs.

This work describes the synthesis and characterization of a pomalidomide-based, bifunctional homo-PROTAC as a novel approach to induce ubiquitination and degradation of the E3 ubiquitin ligase cereblon (CRBN), the target of thalidomide analogs.

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple ...

Chemistry

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.
This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including&#160;apoptosis,&#160;pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and&#160;cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and&#160;leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases,&#160;from other cell death pathways.
Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...