Tissue from a deceased patient with COVID-19 pneumonia and invasive ventilation was obtained postmortem. Immunohistochemistry for Iba1 revealed many microglia nodules within the medulla of this patient. Tissues were manually dissociated and filtered using a 40μm cell strainer. Mononuclear cells were isolated using density gradient centrifugation. Viable cells were counted and stained for mass cytometry. Mass cytometry reagents were obtained or generated by custom conjugation to isotope-loaded polymers using the Maxpar X8 antibody labeling kit. Mass cytometry antibodies used are shown in Table S3 . Staining was performed after β2-microglobulin-based sample barcoding. Briefly, single-cell suspensions were pelleted, incubated with 20 μM Lanthanum-139-loaded maleimido-mono-amine-DOTA in PBS for 10min at RT for live/dead discrimination (LD). Cells were washed in staining buffer and resuspended in surface antibody cocktail, incubated for 30min at RT, washed twice in staining buffer, fixed and permeabilized using FoxP3 staining buffer set, and stained intracellularly for 60min at RT. Cells were further washed twice before fixation in 1.6% PFA solution containing 125nM Iridium overnight at 4°C. Prior to data acquisition on CyTOF Helios (Fluidigm), cells were washed twice in PBS and once in cell acquisition solution.
Helios cytof
Manufactured by Standard BioTools
Sourced in Cameroon
The Helios CyTOF is a mass cytometry instrument developed by Standard BioTools. It is designed to analyze the expression of multiple proteins simultaneously in single cells, providing detailed insights into the cellular composition and function of complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Eq four element calibration beads, by Standard BioTools (2 mentions)
Cell id intercalator ir, by Standard BioTools (1 mentions)
Cell id cisplatin 195pt, by Standard BioTools (1 mentions)
Maxpar cell staining buffer, by Standard BioTools (1 mentions)
Cell id cisplatin 198pt, by Standard BioTools (1 mentions)
40 μm cell strainer, by Avantor (1 mentions)
Foxp3 fixation buffer, by Thermo Fisher Scientific (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Cytof software, by Standard BioTools (1 mentions)
Eq calibration beads, by Standard BioTools (1 mentions)
Milli q, by Merck Group (1 mentions)
Cisplatin, by Standard BioTools (1 mentions)
11 protocols using helios cytof
1
Postmortem Analysis of COVID-19 Microglia
2
CyTOF-based Immune Cell Phenotyping
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Samples were processed as previously described.51 After thawing, cells were classified as unstimulated and stimulated. After an overnight rest, stimulated cells were incubated with phorbol myristate acetate (PMA)+ionomycin; during incubation, anti-CD107a, brefeldin A, and monensin were added to all samples. Dead cells were identified using Cell-ID Cisplatin-195Pt (Fluidigm) prior to surface staining and fixed with 2% paraformaldehyde, followed by intracellular and DNA staining with Cell-ID Intercalator-Ir (Fluidigm) (antibodies are provided in supplemental Tables 2 and 3, available on the Blood Web site). Prior to acquisition (CyTOF Helios; Fluidigm), cells were washed with Milli-Q water and resuspended in a 1× solution of EQ Four Element Calibration Beads (Fluidigm). Data were normalized using MATLAB normalizer before analyzing with Cytobank.52
3
Multiparameter Profiling of Immune Cells
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Cryopreserved LPMC and PBMC were barcoded with a combination of anti-CD45 antibodies (89Y, 115In, and 175Lu) for 30 min at RT and washed with Maxpar Cell Staining Buffer (Fluidigm). After centrifugation, cells were stained with an antibody mix (SI Appendix, Tables S1 and S5 ) for 45 min at RT. After washes, cells were incubated with cisplatin (1:1,000, Cell-ID™ Cisplatin-198Pt; Fluidigm) for 5 min at RT, quenched with Maxpar Cell Staining Buffer, and fixed with 2% PFA for 30 min at 4 °C. For intracellular staining, eBioScience Fix/Perm Buffer (Invitrogen) was added to cells and centrifuged, stained with markers dissolved in Fix/Perm Buffer for 45 min at 4 °C, and washed with Fix/Perm Buffer. After centrifugation, the pellet was incubated with iridium (1:1,000, Cell-ID™ Intracalator-Ir; Fluidigm) overnight at 4 °C, washed, and acquired on CyTOF Helios (Fluidigm). EQ beads were used for normalization.
4
CyTOF Acquisition of Cell Samples
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Prior to acquisition, cells were washed twice with Milli-Q water and resuspended in a 1:10 dilution of EQ Four Element Calibration Beads [Fluidigm] to a concentration of 0.5 × 106 cells/mL. Samples were acquired using a CyTOF Helios [Fluidigm] and data normalized to mass bead signal using the Nolan lab Matlab software [GitHub, https://github.com/nolanlab ]. Each Barcode set was fully acquired within 1 day of mass cytometry run time, and all barcode sets were acquired on sequential days.
5
Human Lymph Node Immune Profiling
6
Multiparametric Profiling of Immune Cells
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Spleen and lung cells from mice, and human PBMCs were first stained with 200 µM cisplatin (Sigma) for the discrimination of dead cells. Upon this, cells were washed and stained with a primary antibodies cocktail for 30 minutes at 4 °C (Supplementary Table 1 (for human antibodies) and Supplementary Table 5 (for mouse antibodies)). Dilution of different antibodies used was either 1:100 or 1:200 and was standardized by our CyTOF platform. After washing, cells were stained with the heavy metal-labeled antibodies cocktail for 30 minutes at 4 °C. Cells were washed, fixed and permeabilised using fixation FoxP3 buffer (eBioscience) for 45 minutes at 4 °C. Upon this cells were stained with intranuclear antibodies cocktail for 30 minutes at 4 °C. Labeled cells were washed twice with PBS and fixed in 2% or 4% PFA overnight. Subsequently, cells were washed and stained for barcodes (30 minutes at 4 °C) and DNA (10 minutes at 4 °C). Samples were acquired on the Helios CyTOF (Fluidigm), and analyzed as mentioned before24 ,25 (link),71 (link). For details, see supplementary methods .
7
Mass Cytometry Sample Preparation
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On the day of sample acquisition, the cells were diluted in 1 ml FACS buffer and washed. The cells were then washed twice in MilliQ water, and then re-suspended at 1 × 106/ml in 1:10 EQ calibration beads (Fluidigm). A minimum number of 500,000 events were then acquired on a mass cytometer (Helios CyTOF, Fluidigm) using CyTOF software (Fluidigm). Following acquisition, all flow cytometry standard (.fcs) files were normalised to bead signal levels using CyTOF software (see [41 (link)] for more details).
8
CyTOF Analysis of Immune Cell Profiles
9
Mass Cytometry Time Series Standardization
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To minimize the effect of experimental variability on mass cytometry measurements between serially collected samples, samples corresponding to the entire time series collected from one woman were processed, barcoded, pooled, stained, and ran simultaneously. To minimize the effect of variability between study participants, the run was completed within consecutive days, while carefully controlling for consistent tuning parameters of the mass cytometry instrument (Helios CyTOF, Fluidigm Inc., South San Francisco, CA).
10
Mass Cytometry Variability Minimization
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To minimize the effect of experimental variability on mass cytometry measurements between serially collected samples, samples corresponding to the entire time series collected from one participant were processed, barcoded, pooled, stained, and run simultaneously. To minimize the effect of variability between study participants, samples sets of patients matched for control and treatment (HSR dataset), or randomized for time of sampling (NTP and LSR datasets). Further, the run was completed within consecutive days, while carefully controlling for consistent tuning parameters of the mass cytometry instrument (Helios CyTOF, Fluidigm Inc., South San Francisco, CA).
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