Name: Mapping Infant Immunity with Minimal Input: Integrative Single-Cell and Multiomic Profiling (Video) | JoVE Journal
Uploaded: 2026-04-03T12:35:00.000+00:00
Duration: 10 min 29 s
Description: Learn high-dimensional multiomic immune profiling from minimal neonatal blood samples using flow cytometry, proteomics, and single-cell RNA sequencing. This protocol enables longitudinal studies of early-life immunity in preterm and term infants.

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5 minutes

We thank the following core facilities at Yale: Yale Flow Cytometry core; Yale Center for Genome Analysis. We thank the Yale Center for Clinical Investigation, the Yale Office of Physician-Scientist and Scientist Development, and the Pediatric Critical Care and Trauma Scientist Development Program/NICHD. Respirometry studies were performed by the Chemical Metabolism Core at Yale University. This work was funded by grants to: P01 AI179570 (L.K.), R01AI171980 (L.K.), R01DK129552 (L.K.), R01HL163043 (L.K.), Cystic Fibrosis Foundation (L.K.); K08 AI177743 (N.N.B.), Hartwell award (N.N.B.); K08DK133687 (O.O.), Yale School of Medicine, Department of Paediatrics (O.O.); R01AI123204 (C.R.O). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Figure 1 was created in BioRender. Gawon, K. (2025)&#160;https://BioRender.com/7embw10.

Informed parental consent and full compliance with relevant national ethical regulations were ensured in accordance with Yale University&#39;s Institutional Review Board (IRB). As specified in the approved IRB protocol, parents were contacted by the research team during the first week following the infant&#39;s birth to obtain consent prior to enrollment. No study procedures involving human-derived materials commenced prior to documented ethics approval.
1. Sample collection, processing, and storage (Figure 1)
<ol>
	<li>Inpatient blood collection
	<ol>
		<li>Collect and process infant samples after receiving them, store at 4 &#176;C, and process within 12 h of collection.</li>
		<li>Collect ~250 &#181;L of whole blood and store in an anticoagulant EDTA blood collection tube. Invert the blood collection tube 8x to mix the anticoagulant.</li>
		<li>Proceed to process the whole blood via red blood lysis as described in Section 1.3 (whole blood sample processing).</li>
		<li>To collect dry blood spots, use the sterile lancet to make a puncture on the cleaned toe/heel.
		<ol>
			<li>Alternatively, pipette ~25-30 &#181;L of blood from the EDTA tube onto the filter paper. Lightly touch the filter paper to the blood drop.</li>
		</ol>
		</li>
		<li>Fill at least one circle with free-flowing blood, one drop per circle. Allow the blood spot to dry. Keep the filter paper open until the blood spots are dry thoroughly.</li>
		<li>Label appropriately with time of collection, processing date, timepoint, and specimen Identification.</li>
		<li>Place in a clear plastic zipper storage bag. Store blood spot cards at &#8722;20 &#176;C or &#8722;80 &#176;C for extended stability until ready for proteomic analysis.</li>
	</ol>
	</li>
	<li>Outpatient blood collection 
	&#8203;NOTE: With approval from Yale University&#39;s Institutional Review Board, parents of outpatient participants were given the option to collect blood samples at home using a microneedle self-collection device. This device obtains capillary blood through a lancet applied to the skin. Parents who selected this method received in-clinic training using demonstration kits and were subsequently provided with detailed written instructions and home-use collection kits.
	<ol>
		<li>Wash hands and wear gloves before use. Collect all necessary items, including a mirror if only one parent is performing the blood collection.</li>
		<li>Remove the device from the tool kit and twist off the cap from a compatible tube.</li>
		<li>Press the tube into the device until snug with fill lines facing out.</li>
		<li>Remove the infant&#8217;s diaper and hold the infant upright.</li>
		<li>Follow the heat pack&#39;s instructions to activate. 
		&#8203;NOTE: If a pack is not available, use some other warming method, such as a hand.</li>
		<li>Hold the heat pack on the site (buttock) for 2-4 min to warm and increase blood flow.</li>
		<li>Clean the area with an alcohol pad and allow to dry.</li>
		<li>Remove the clear cover from the red button. Peel the tab from the device and firmly stick onto the infant&#39;s fullest part of the buttocks while holding them upright. 
		&#8203;NOTE: Adjust their posture as needed to keep the tube completely vertical for proper blood collection.</li>
		<li>Press the button all the way down once and release. Set the timer for 5 min. Watch the tube fill using the mirror as needed.</li>
		<li>Blood may not appear for the first 1-2 min. Remove the device after a maximum of 5 total min or sooner if blood fills to the top indicator line before 5 min.</li>
		<li>Remove the tube from the device with a slight twist and pull down. Quickly snap the cap fully unto the tube. Invert the tube at least 10x, ensuring the blood touches the cap with each inversion to prevent clotting.</li>
		<li>Ship samples overnight after collection.</li>
	</ol>
	</li>
	<li>Whole blood sample processing
	<ol>
		<li>Process blood samples in a biological safety cabinet.</li>
		<li>Carefully transfer the 250 &#181;L of blood into a 50 mL conical tube containing 10 mL of 1x RBC lysis buffer. Rinse the blood collection tube with 10 mL of RBC lysis buffer until empty. Allow the cells to lyse at room temperature (RT) for 15 min.</li>
		<li>Once lysis is complete, add ~30 mL of PBS at RT to wash and spin at 430 &#215; g for 5 min at 4 &#176;C. Aspirate the supernatant using a vacuum, without disturbing the pellet.</li>
		<li>Continue transferring and rinsing the sample as outlined in step 1.3.2 until the cell pellet appears free of visible red blood cells. Centrifuge at 430 &#215; g for 5 min, then carefully remove the supernatant without disturbing the pellet.</li>
		<li>For every 250 &#181;L of blood collected, resuspend the resulting cell pellet in 2 mL of freezing media (10% DMSO in FBS) and split into two cryovials for preservation.</li>
		<li>Check cell viability by counting cells resuspended in feezing media with a hemocytometer using trypan blue exclusion.</li>
		<li>Transfer 1 mL from step 1.3.5 to each cryovial. Label appropriately with time of collection, processing date, time point, and specimen Identification.</li>
		<li>Store cryovial(s) intermediately at -80 &#176;C in a freezing container with isopropanol. After 24 h, transfer the cryovial(s) to liquid nitrogen until ready for analysis.</li>
	</ol>
	</li>
</ol>
2. TMPA from dried blood spots (DBS) (Figure 2)
<ol>
	<li>On the day of processing, bring the filter paper to RT. Using a 3 mm metal puncher, perforate 2-3 circles in the filter paper where the blood went through, as centrally on the blood spot as possible.</li>
	<li>Pick up each 3 mm punch with tweezers and add to a well of a 96-well plate or a low-binding PCR tube containing 20 &#181;L of elution buffer (1x PBS, 1x Protease Inhibitor, 0.05% Tween 20). Make sure the punch is covered by elution buffer.</li>
	<li>Close the tubes or cover the plate with a plastic skirt, and place them on a Digital Vortex rotating at 600 rpm for 60 min at RT.</li>
	<li>At the end of incubation, carefully transfer the eluate to a new low-binding tube or a 96-well plate. Discard the tubes or plate with the filter punches.</li>
	<li>Quantitate the protein content using BCA assay.
	<ol>
		<li>Take 2 &#181;L of each sample or BSA standard to make serial dilutions of 1:10 and 1:100 in water. Prepare samples and standards in duplicate by adding 15 &#181;L of diluted samples or standard to 200 &#181;L of 1x working solution. Incubate samples for 30 min at 37 &#176;C.</li>
		<li>Read the signal at 562 nm on a plate reader. Calculate the final protein concentration based on the BSA standard slope and elution buffer background.</li>
	</ol>
	</li>
	<li>Make aliquots of 30 &#181;L for samples at 0.5-1 mg/mL in low-binding 1.5 mL screw cap micro tubes. Freeze samples at -80 &#176;C for later submission to be processed using TMPA service.</li>
</ol>
3. Spectral flow cytometry of RBC lysed blood (Figure 3)
<ol>
	<li>On the day of the experiment, thaw a vial of RBC lysed cells quickly in a 37 &#176;C water bath until one small drop of ice remains.</li>
	<li>Add the cell suspension to a 15 mL conical tube containing 10 mL of prewarmed (37 &#176;C) complete RPMI (prepared using 500 mL of RPMI 1640, 50 mL of FBS, 5.6 mL of Penicillin-Streptomycin, and 5.6 mL of glutamine supplement). Centrifuge at 400 &#215; g for 5 min at room temperature. Carefully remove and discard the supernatant.</li>
	<li>Gently resuspend the pellet in 3 mL of complete RPMI and determine cell count using an appropriate method.</li>
	<li>Spin the resuspended cells at 400 &#215; g for 5 min at room temperature. Aspirate and discard the supernatant.</li>
	<li>&#160;Adjust the cell concentration to 1 &#215; 106&#160;cells/mL in complete RPMI. Dispense 100 &#181;L per well (0.1 &#215; 10&#8310; cells) into a 96-well V-bottom plate. Centrifuge at 764 &#215; g for 2 min at 4 &#176;C and remove the supernatant.</li>
	<li>Add 100 &#181;L of Live/Dead dye stock in PBS at a 1:2,000 dilution with Fc block and monocyte block (1:100 dilution) to all samples, except the unstained control. Incubate for 15 min at 4 &#176;C.</li>
	<li>Wash with 100 &#181;L of PBS and centrifuge at 764 &#215; g for 2 min at 4 &#176;C. Carefully discard the supernatant. 
	NOTE: It is imperative to use Fc block and monocyte block to prevent any non-specific binding of the antibodies.</li>
	<li>Add 100 &#181;L of the surface cocktail diluted (1:200 dilution) in FACS buffer (500 mL of PBS, 2.5 g of bovine serum albumin (BSA), 2 mM EDTA, pH 8.5) to all samples, except the unstained control. Incubate for 30 min at 4 &#176;C.</li>
	<li>Wash with 100 &#181;L of FACS buffer and centrifuge at 764 &#215; g for 2 min at 4 &#176;C. Carefully discard the supernatant.</li>
	<li>Fix the cells by adding 100 &#181;L of 4% paraformaldehyde to each sample. Incubate for 15 min at RT in the dark.</li>
	<li>Wash with 100 &#181;L of FACS buffer, centrifuge at 764 &#215; g for 2 min at 4&#176;C, and resuspend in 200 &#181;L of FACS buffer.</li>
	<li>Prepare ultra-comp beads as single-color controls. Stain the single-color control beads using the same protocol as the samples, including fixation if the samples were fixed.</li>
	<li>Prepare Fluorescent Minus One (FMO) controls using the same procedure as for the samples. 
	NOTE: It is important to treat the single-color controls and the FMOs in the same way as the samples. For example, if the samples are fixed after staining, the FMOs and the single-color controls should be fixed as well.</li>
	<li>Run the samples, single-color controls, unstained controls, and FMO controls on a conventional/spectral flow cytometer. Analyze the data using software.</li>
	<li>Set gates based on appropriate isotype and FMO controls. First, gate cells to select the leukocyte population using the forward scatter (FSC) versus side scatter (SSC) plot, which helps exclude debris and select cells based on size and granularity.</li>
	<li>Following leukocyte selection, gate cells to exclude doublets using a plot of forward scatter area (FSC-A) versus forward scatter height (FSC-H). 
	NOTE: This ensures the selection of single cells (singlets), with cells falling along the diagonal considered as singlets, while doublets and clumps are excluded.</li>
	<li>Gate cells for viability using a viability dye to exclude dead cells, ensuring that the subsequent analysis includes only live cells.</li>
	<li>After these initial gating steps, further gate live single cells for specific lineage markers to subcategorize different immune cell populations. Divide the live single cells into CD33+ myeloid cells and CD33- lymphocytes based on CD33 expression.</li>
	<li>Within the CD33+ population, differentiate CD14+ monocytes and identify CD11c+ dendritic cells (DCs) as cells expressing CD11c but not CD14.</li>
	<li>Within the CD33- lymphocyte population, gate CD3+ T cells and sub-gate to identify CD4+ and CD8+ T cell subsets.</li>
	<li>Additionally, identify CD56+ natural killer (NK) cells by CD56 expression, and gate CD19+ B cells by CD19 expression.</li>
</ol>
4. scRNA-seq of RBC lysed blood (Figure 4)
<ol>
	<li>Thaw one vial of cryopreserved RBC lysed cells in a 37 &#176;C water bath. Add the sample to 10 mL of T-cell media in a 15 mL conical tube. Centrifuge at 430 &#215; g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Add another 10 mL of 1x PBS to the sample. Centrifuge at 430 &#215; g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Count the cells and assess viability with trypan blue. If viability is less than 70%, proceed to dead cell removal with Dead Cell Removal Kit.
	<ol>
		<li>Dead cell removal
		<ol>
			<li>Dilute the 20x Binding Buffer Stock using sterile, double-distilled water to obtain a 1x working solution.</li>
			<li>Suspend the cell pellet in 100 &#181;L of dead cell removal MicroBeads per 1 &#215; 107&#160;total cells. Mix thoroughly and incubate for 15 min at room temperature (RT).</li>
			<li>Insert the magnetic separation column into the appropriate magnetic stand. Rinse the column with 500 &#181;L of 1x Binding Buffer to equilibrate before use.</li>
			<li>Bring the cell volume to 500 &#181;L with 1x binding buffer. Apply the cell suspension to the column and collect the flowthrough in a new 1.7 mL microcentrifuge tube.</li>
			<li>Wash 2x with 500 &#181;L of binding buffer. Collect the flowthrough in the same microcentrifuge tube.</li>
			<li>Centrifuge the eluate at 300 &#215; g for 10&#160;min. Discard the supernatant.</li>
		</ol>
		</li>
		<li>Count the cells and assess their viability with trypan blue.</li>
		<li>Resuspend 20,000 viable cells in 40 &#181;L of PBS with 0.04% BSA in a new 1.7 mL microcentrifuge tube.</li>
		<li>Send the cells on ice immediately to a genomic core facility for library generation and sequencing. Collect the sequencing results in the format of Cell Ranger outputs.</li>
	</ol>
	</li>
</ol>

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<li>Daae, L. N. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+haematological+parameters+in+capillary+and+venous+blood+samples+from+hospitalized+children%5BTitle%5D)+AND+%22Scand+J+Clin+Lab+Invest%22%5BJournal%5D)">Comparison of haematological parameters in capillary and venous blood samples from hospitalized children.</a> Scand J Clin Lab Invest. 51 (7), 651-654 (1991).</li>
<li>Meites, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skin-puncture+and+blood-collecting+technique+for+infants%5BTitle%5D)+AND+%22Clin+Chem%22%5BJournal%5D)">Skin-puncture and blood-collecting technique for infants.</a> Clin Chem. 34 (9), 1890-1894 (1988).</li>
<li>Grüner, N., Stambouli, O., Ross, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dried+blood+spots%3A+preparing+and+processing%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Dried blood spots: preparing and processing.</a> J Vis Exp. (97), e52619(2015).</li>
<li>Waaijer, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spectral+flow+cytometry+panel+for+comprehensive+immunophenotyping%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Spectral flow cytometry panel for comprehensive immunophenotyping.</a> Cytometry A. 107 (4), 226-232 (2025).</li>
<li>Thieme-Ehlert, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((OMIP-114%3A+a+36-color+spectral+flow+cytometry+panel%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">OMIP-114: a 36-color spectral flow cytometry panel.</a> Cytometry A. 107 (6), 364-371 (2025).</li>
<li>Yamawaki, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+comparison+of+high-throughput+single-cell+RNA-seq+methods%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Systematic comparison of high-throughput single-cell RNA-seq methods.</a> BMC Genomics. 22, 66(2021).</li>
<li>Stoeckius, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+epitope+and+transcriptome+measurement+in+single+cells%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Simultaneous epitope and transcriptome measurement in single cells.</a> Nat Methods. 14 (9), 865-868 (2017).</li>
<li>Grandi, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+accessibility+profiling+by+ATAC-seq%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Chromatin accessibility profiling by ATAC-seq.</a> Nat Protoc. 17 (6), 1518-1552 (2022).</li>
<li>Kim, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perspectives+on+single-nucleus+RNA+sequencing%5BTitle%5D)+AND+%22J+Pathol+Transl+Med%22%5BJournal%5D)">Perspectives on single-nucleus RNA sequencing.</a> J Pathol Transl Med. 57 (1), 52-59 (2023).</li>
</ol>

The authors declare that they have no conflicts of interest.

Obtaining high-quality immunological data from minimal volumes of neonatal and infant blood requires methodological adaptations across multiple steps. In this protocol, we describe four complementary strategies: (i) optimized blood collection and cryopreservation to preserve viable cells for downstream use, (ii) application of spectral flow cytometry for multiparametric immunophenotyping, (iii) scRNA-seq to resolve immune heterogeneity at high resolution, and (iv) TMPA. Together, these approaches maximize data yield whil...

This protocol is optimized for 100-250 &#181;L blood samples processed within 12 h of collection, either cryopreserved or used immediately. At birth, the neonatal immune system is thrust into a radically different environment, enriched with microbial and environmental stimuli that were previously absent in the protected intrauterine setting1,2. This shift requires the immune system to adapt and respond to a vast array of new antigens, a process that is essential for survival but also poses significant challenges1,2. Increasing evidence suggests that early-life immunity is not simply immature or a miniature version of the adult immune system. Instead, it is uniquely tailored to meet the specific needs of this critical developmental period3,4. During this neonatal window, the immune system&#39;s cellular components, including various types of T cells, B cells, and myeloid cells, mature and develop unique characteristics and functions specific to early life5,6,7,8. Despite its importance, neonatal immunity remains difficult to study directly due to ethical and practical constraints. A major limitation is the small blood volume in neonates, averaging ~100 mL/kg9,10. This challenge becomes even more pronounced when collecting blood from extremely premature infants (born before 30 weeks of gestation) and ELBW infants, who can weigh as little as 400 g with only 40 mL of circulating blood. This restricts how much blood can be safely collected, underscoring the need for approaches that maximize data yield from minimal samples. To address this challenge, our work demonstrates optimized protocols for high-dimensional immune profiling using minute blood volumes (100-250 &#181;L), such as those obtained from ELBW infants.
Recent advances in blood collection and processing have enabled the study of immune responses in populations where sample acquisition is traditionally difficult. Microneedle devices such as the Tasso and Touch Activated Phlebotomy systems were initially developed for convenient at-home blood collection in adults. They have since been adapted for use in infants, allowing primary caregivers and clinical teams to collect samples with minimal discomfort. Additionally, they can be used at home without the stress of repeated clinical visits11, facilitating compliance and accessibility. An ethylenediaminetetraacetic acid (EDTA) microtube should be used with the device and shipped overnight to the lab with adherence to appropriate biohazard protocols. The next morning, when samples arrive, they should be processed using red blood cell (RBC) lysis methods to capture circulating immune cells6. Of note, EDTA tubes enable live-cell isolation, but time considerations are important. For functional assays with stimulation, samples must be processed within less than 4 h and ideally less than 2 h from collection. For the non-functional, phenotypic assays presented below, samples should be processed within 12 h to permit overnight shipment while ensuring recovery, as cell viability decreases over time12. Compared to traditional density-gradient separation with Ficoll, lysis is faster, less labor-intensive, and yields higher recovery13. However, peripheral blood mononuclear cell isolation by lysis may leave contaminating granulocytes behind, reducing purity but can be advantageous for studies interested in these cell types14. These tradeoffs highlight how methodological adaptations balance feasibility and accuracy when studying rare or sensitive clinical samples, such as those from neonates. Downstream handling of isolated cells has also benefited from methodological refinements. Cryopreservation with fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) remains a robust approach for immune cells, particularly in fetal and neonatal blood, where cell viability and recovery are critical. Alongside optimizing the blood collection process, we have also adopted downstream techniques that allow for the extraction of maximum information from small sample volumes. We introduce a set of optimized protocols that facilitate sampling across early life, enabling a detailed examination of immune dynamics over time. Our approach leverages advanced techniques such as spectral flow cytometry, proteomics, and single-cell RNA sequencing, which collectively offer an unparalleled view of the proteomic and transcriptional signatures of circulating immune cells in early life. Additionally, spectral flow cytometry offers opportunities to gain functional insight with minimal cell input.
Over the years, several techniques, such as complete blood counts (CBCs), have been used to analyze blood-derived immune cells15, while others have analyzed immune cell ratios in neonatal whole blood samples using DNA methylation16,17. While these techniques have been helpful for learning the proportions of known immune cells, there are still limitations in our ability to gain in-depth insights into cells without a multi-omics system. CBCs provide only broad measures of major blood cell types, making them useful for establishing overall immune status in a clinical setting but limited in their ability to resolve subtypes or functional states. Most recently, studies have begun to address this limitation by employing plasma-derived transcriptomic and metabolomic changes in infants as early as the first week of life18. Mass spectrometry-based proteomics has been adapted to study immune profiles in neonatal samples19. This approach is powerful for measuring a broad range of proteins in an unbiased manner and for identifying potential biomarkers. However, key limitations include the requirement for relatively large protein input and the lack of direct information on cell identity, despite capturing both soluble and insoluble proteins. Targeted multiplex proteomics assay (TMPA) offers alternatives for low-input samples. These methods use targeted antibody-based detection, providing high-sensitivity profiling for samples with limited material. One of these, in particular, has been used to track early-life immune development trajectories longitudinally20.
Flow cytometry enables quantitative analysis of immune cell proteins at the single-cell level by passing fluorescently labeled cells through a focused laser beam and measuring the resulting light scatter and fluorescence signals. In this technique, fluorescently labeled antibodies bind to specific surface or intracellular proteins. This allows for simultaneous detection and quantification of multiple immune cell subtypes and their defining characteristics based on their fluorescence profiles. Spectral flow cytometry extends this approach by capturing the full emission spectrum of each fluorochrome, enabling precise spectral unmixing and the simultaneous detection of over 35 markers in a single assay21,22,23. This enables analysis of many more markers than conventional flow cytometry panels, which are limited to ~8-12 markers. Complementing these cytometric techniques, single-cell RNA sequencing (scRNA-seq) has transformed immunological studies by enabling high-resolution, unbiased transcriptomic profiling from minimal input material24. Despite higher costs and dependence on cell viability, scRNA-seq provides unparalleled insights into early-life immune heterogeneity compared to bulk sequencing. Collectively, these advances demonstrate how innovations in collection, preservation, and analysis are being adapted to overcome the unique challenges of studying fetal and neonatal immunity.
This protocol has been optimized for the analysis of cryopreserved peripheral immune cells isolated via serial RBC lysis. Researchers should consider this protocol if their studies require high-resolution immune profiling from small volumes of blood (100-500 &#181;L), especially when longitudinal analysis is critical. The method is broadly applicable to both term and preterm neonates and is compatible with a wide range of downstream analyses, making it suitable for diverse research questions in neonatal and pediatric immunology.

<table><tbody><tr><td>0.5 M EDTA, pH 8.0</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>15575-038&amp;nbsp;</td><td></td></tr><tr><td>1x RBC Lysis Buffer&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>3177165&amp;nbsp;</td><td></td></tr><tr><td>BCA Protein Assay Kit &amp;nbsp;</td><td>Abcam&amp;nbsp;</td><td>ab287853&amp;nbsp;</td><td></td></tr><tr><td>BD Microtainer MAP Microtube</td><td>Fisher Scientific&amp;nbsp;</td><td>22-253-270</td><td></td></tr><tr><td>Bel-Art Cryo-Safe -1&amp;deg;C Freeze Controller&amp;nbsp;</td><td>Millipore Sigma&amp;nbsp;</td><td>41122800&amp;nbsp;</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A9647-100G&amp;nbsp;</td><td></td></tr><tr><td>Brilliant Ultra Violet 496 Anti-Human CD3&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>364-0038-42&amp;nbsp;</td><td></td></tr><tr><td>Brilliant Ultra Violet 661 anti-Human CD11c&amp;nbsp;</td><td>BD BioScience&amp;nbsp;</td><td>612968&amp;nbsp;</td><td></td></tr><tr><td>Brilliant Vil750 Anti-Human CD14&amp;nbsp;</td><td>BioLegend&amp;nbsp;</td><td>367135&amp;nbsp;</td><td></td></tr><tr><td>Brilliant Violet 570 Anti-Human CD8a&amp;nbsp;</td><td>BioLegend&amp;nbsp;</td><td>301038&amp;nbsp;</td><td></td></tr><tr><td>Brilliant Violet 711 Anti-Human CD56&amp;nbsp;</td><td>BioLegend&amp;nbsp;</td><td>318336&amp;nbsp;</td><td></td></tr><tr><td>cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td><td>Millipore Sigma&amp;nbsp;</td><td>11836170001&amp;nbsp;</td><td></td></tr><tr><td>Dead Cell Removal Kit&amp;nbsp;</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-090-101&amp;nbsp;</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>BP231-100&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate buffered saline (DPBS), no calcium, no magnesium&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>14190144&amp;nbsp;</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>A5256801&amp;nbsp;</td><td></td></tr><tr><td>Fisherbrand Digital Vortex Mixer&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>02215418&amp;nbsp;</td><td></td></tr><tr><td>Fixation Buffer&amp;nbsp;</td><td>BioLegend&amp;nbsp;</td><td>420801&amp;nbsp;</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco&amp;nbsp;</td><td>35050061&amp;nbsp;</td><td></td></tr><tr><td>MACS MultiStand&amp;nbsp;</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-042-303&amp;nbsp;</td><td></td></tr><tr><td>MS columns&amp;nbsp;</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-042-201&amp;nbsp;</td><td></td></tr><tr><td>Name</td><td>Company</td><td>Catalog Number</td><td></td></tr><tr><td>OctoMACS Separator&amp;nbsp;</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-042-109&amp;nbsp;</td><td></td></tr><tr><td>Pacific Blue Anti-Human CD4&amp;nbsp;</td><td>BioLegend&amp;nbsp;</td><td>344619&amp;nbsp;</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>15140-122&amp;nbsp;</td><td></td></tr><tr><td>RB780 Anti-Human CD33&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>755606&amp;nbsp;</td><td></td></tr><tr><td>Rierdge 3mm Metal Puncher&amp;nbsp;</td><td>Rierdge&amp;nbsp;</td><td>sdd231031pc-01&amp;nbsp;</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Gibco&amp;nbsp;</td><td>11875-093&amp;nbsp;</td><td></td></tr><tr><td>Spark UV 387 Anti-Human CD19&amp;nbsp;</td><td>BioLegend&amp;nbsp;</td><td>302289&amp;nbsp;</td><td></td></tr><tr><td>Tasso Collection Kits</td><td>Tasso&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>Thermo Scientific Screw Cap Micro Tubes</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>14-755-287&amp;nbsp;</td><td></td></tr><tr><td>Whatman 309 Proteinsaver</td><td>Cytiva&amp;nbsp;</td><td>41111700&amp;nbsp;</td><td></td></tr><tr><td>Zombie NIR Fixable Viability Kit&amp;nbsp;</td><td>BioLegend&amp;nbsp;</td><td>423105&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Software and packages&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adobe Illustrator</td><td>2024</td><td></td><td></td></tr><tr><td>bbknn</td><td>1.5.1</td><td></td><td></td></tr><tr><td>FlowJo</td><td>10.8.1</td><td></td><td></td></tr><tr><td>GraphPad Prism</td><td>9.4.0</td><td></td><td></td></tr><tr><td>Name</td><td>Version</td><td></td><td></td></tr><tr><td>Scanpy</td><td>1.9.1</td><td></td><td></td></tr><tr><td>Srublet</td><td>0.2.3</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Commercial assays&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chromium Single Cell 5&amp;rsquo; Kit</td><td>10X Genomics</td><td>PN-1000263</td><td></td></tr><tr><td>Olink&amp;nbsp;Reveal or Target 96 Inflammatory panel</td><td>Olink</td><td>https://olink.com/products/olink-target-96</td><td></td></tr></tbody></table>

mapping infant immunity with minimal input: integrative single-cell and multiomic profiling

Mapping Infant Immunity with Minimal Input: Integrative Single-Cell and Multiomic Profiling

At birth, the neonatal immune system is abruptly confronted with a radically different environment, rich in microbial and environmental stimuli. Increasing evidence suggests that early-life immunity is not merely immature, but rather uniquely adapted to meet the distinct demands of this developmental window. During this period, the immune system is highly dynamic, undergoing rapid changes to accommodate the shifting external landscape. Understanding this complex and evolving immune landscape requires direct study in neonates. However, a major limitation in such investigations is the low total blood volume in neonates (~100 mL/kg), which restricts the amount that can be safely collected for research, particularly in extremely low birth weight (ELBW) or extremely premature infants. In our recent work, we address this limitation by demonstrating how high-dimensional immune profiling can be performed using minute volumes of neonatal blood. We introduce a set of optimized protocols for longitudinal sampling across early life and apply advanced techniques, including flow cytometry, proteomics, and single-cell RNA sequencing, to maximize the information obtained from minimal input. Together, these methods enable a comprehensive view of the transcriptomic and proteomic signatures of circulating immune cells in neonates, offering new insights into the unique trajectory of early-life immunity.

We collected DBS cards from 10 extremely preterm infants at three time points: 1 week (n = 12), 1 month (n = 7), and 2 months (n = 3) for TMPA proteomic analysis6 (Figure 2A). These samples were analyzed alongside full-term infant cord blood (TCB; n = 4) and healthy adult blood (AB; n = 5). The normalized protein expression (NPX) of 92 immune-related proteins was quantified using the TMPA platform (Figure 2B). Statistical analysis demonst...

This protocol describes the collection and preservation of blood samples from neonatal and pediatric patients and the application of scRNA-seq, proteomics, and spectral flow cytometry to characterize the immune cell populations from these samples.

At birth, the neonatal immune system is abruptly confronted with a radically different environment, rich in microbial and environmental stimuli. ...

Our research defines how early-life immunity adapts after birth using longitudinal high-dimensional multiomic profiling from small blood volumes. This protocol can be applied to neonatal and pediatric studies for longitudinal immune profiling from limited biospecimens across health, infection and inflammation. To begin, quickly thaw a vial containing lysed infant red blood cells in a 37-degrees-Celsius water bath.Transfer the thawed cells to a 15-milliliter conical tube containing 10 milliliters of warm complete RPMI medium at 37 degrees Celsius. Centrifuge the tube at 400 g for five minutes at room temperature, and discard the supernatant. Re-suspend the cell pellet in three milliliters of complete RPMI medium and count the total number of cells.Dilute the sample if the concentration is too high to count. After counting the cells, repeat the centrifugation step and re-suspend the cells in complete RPMI medium to obtain a concentration of one million cells per milliliter. Transfer 100 microliters containing 0.1 million cells into each well of a 96-well V-bottom plate.Centrifuge the plate at 764 g for two minutes at four degrees Celsius. Discard the supernatant. Next, add 100 microliters of live dead dye stock diluted one to 2, 000 in PBS with FC block and monocyte block diluted one to 100 to all samples except the unstained control.Incubate the samples for 15 minutes at four degrees Celsius. To wash the cells, add 100 microliters of PBS. Centrifuge the plate at 764 g for two minutes at four degrees Celsius, and carefully discard the supernatant.Add 100 microliters of surface antibody cocktail, diluted one to 200 in fluorescence-activated cell sorting, or FACS buffer, to all samples except the unstained control. Incubate for 30 minutes at four degrees Celsius. Then wash the cells with 100 microliters of FACS buffer, centrifuge and discard the supernatant.Fix the cells by adding 100 microliters of 4%paraformaldehyde to each sample. Incubate for 15 minutes at room temperature in the dark, then wash the cells again, and re-suspend in 200 microliters of FACS buffer. Prepare UltraComp compensation beads as single-color controls.Stain the single-color control beads using the same procedure as the samples, including fixation if the samples were fixed. Also, prepare fluorescence minus one or FMO controls in the same manner. Run the samples, single-color controls, unstained controls and fluorescent-minus-one controls on a conventional or spectral flow cytometer.Open FlowJo and select the sample of interest to analyze. Set gates using appropriate isotype and FMO controls. Gate leukocytes using FSC versus SSC to exclude debris, and select cells by size and granularity.Exclude doublets using FSCA versus FSCH. Gate live cells using a viability dye to exclude dead cells. From live single cells, divide into CD33-negative lymphocytes and CD-33-positive myeloid cells.Within CD33-positive cells, identify CD14-positive monocytes. Then within CD33-positive cells, identify CD14-negative, CD11C-positive dendritic cells. Now, among CD33-negative lymphocytes gait CD3-positive T cells and CD3-negative, CD56-positive natural killer cells.Within CD3-positive T cells, identify CD8-positive and CD4-positive subsets and identify CD3-negative, CD56-negative, CD19-positive B cells. To begin, thaw one vial of cryo-preserved red blood cell lysed cells in a 37-degrees-Celsius water bath. Transfer the suspension to a 15-milliliter conical tube containing 10 milliliters of T-cell media and centrifuge at 430 g for five minutes at four degrees Celsius.Discard the supernatant. After re-suspending the cell pellet with one milliliter of PBS, add another nine milliliters of PBS to the sample. Centrifuge the tube at 430 g for five minutes at four degrees Celsius and discard the supernatant.Count the cells and assess viability using trypan blue exclusion. If viability is less than 70%proceed to use a dead cell removal kit as follows. First, prepare 1x-binding buffer by diluting 20x-binding buffer stock solution with sterile double-distilled water.Re-suspend the cell pellet in 100 microliters of dead cell removal microbeads per 10 to the power of seven total cells. Mix well and incubate for 15 minutes at room temperature. Place the magnetic separation column into the appropriate magnetic stand, along with a collection tube.Then equilibrate the column by rinsing with 500 microliters of 1x-binding buffer. Bring the cell suspension volume to 500 microliters with 1x binding buffer. After replacing the collection tube, apply the cell suspension to the column and collect the flow-through in the new 1.7-milliliter microcentrifuge tube.Next, wash the column two times with 500 microliters of binding buffer and collect the flow-through in the same microcentrifuge tube. Centrifuge the eluate at 300 g for 10 minutes and discard the supernatant. Count the cells and assess viability again using trypan blue exclusion.Aliquot 20, 000 viable cells into a new 1.7-milliliter microcentrifuge tube. Then centrifuge the cells at 300 g for 10 minutes and remove the supernatant. Re-suspend the cells with 40 microliters of PBS containing 0.04%BSA.Finally, place the tube containing the cells on ice and send it immediately to a genomic core facility for library generation and sequencing. Collect the sequencing results in the format of cell ranger outputs. Dried blood spot samples from preterm infants and controls were processed.And the normalized protein expression of 92 immune-related proteins was quantified. The expression of lymphocyte-activation gene three and signaling threshold-regulating transmembrane adapter 1 were significantly higher in peripheral blood from preterm infants at two months of age compared with term cord blood. A comprehensive gating strategy was used to identify leukocyte populations from red blood cell lysed white blood cell samples.Within the T-cell population, CD4-positive and CD8-positive subsets were distinguished. After filtering, over 120, 000 cells were captured and profiled in the single-cell ribonucleic acid sequencing dataset. Dimensionality reduction and clustering analysis identified 14 distinct cell clusters.Cell clusters were annotated based on differentially expressed genes and known cell markers. 12 of the 14 clusters corresponded to immune cell lineages, including T cells, natural killer cells, B cells, myeloid cells, and neutrophils. A stacked bar graph illustrated the relative proportion of cells per cluster across one week, one month, and two months of age.This study enables measurement of cellular, transcriptomic, proteomic, and functional immune signatures longitudinally from limited-volume blood samples. Future studies can utilize these methods to define healthy immune trajectories and uncover dysregulated early-life immune responses linked to diseases.

mapping-infant-immunity-with-minimal-input-integrative-single-cell

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Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

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Learn high-dimensional multiomic immune profiling from minimal neonatal blood samples using flow cytometry, proteomics, and single-cell RNA sequencing. This protocol enables longitudinal studies of early-life immunity in preterm and term infants.

Mapping Infant Immunity with Minimal Input: Integrative Single-Cell and Multiomic Profiling

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