Blood plasma from healthy individuals were collected in 6 mL EDTA tubes (Vacuette, 456243) and centrifuged immediately at 3000 rpm at room temperature. Following centrifugation, the plasma was transferred to 0.5 mL tubes (Sarstedt, 72.730.003) and frozen within 20 minutes. The samples were stored at -80°C before being transferred to SciLifeLab for examination. A pool of SIS PrESTs was made and spiked to empty wells of a 96 well LoBind plate (0030129512, Eppendorf, Hamburg, Germany) according to Table 1 . The plate was vacuum centrifuged to dry off any liquid. Ten times diluted plasma corresponding to two microliters of raw plasma was added to the plate of dried standards and the samples were digested following the same procedure as described. Each digest was then subjected to SPE using StageTips as described earlier [48 (link)].
Lobind plate
Manufactured by Eppendorf
Sourced in Germany
LoBind plates are a type of laboratory equipment designed to minimize sample loss. They feature a specialized surface coating that reduces the adsorption of biomolecules, proteins, and other analytes to the plate. This helps preserve sample integrity and concentration during various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Facsaria fusion, by BD (5 mentions)
Brilliant stain buffer, by BD (3 mentions)
Facsaria fusion instrument, by BD (2 mentions)
Cd45 buv395, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Facsaria fusion flow cytometer, by BD (1 mentions)
10 protocols using lobind plate
1
Plasma Proteome Sample Preparation
2
Single-Cell Profiling of Drug-Treated Cells
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For SCP analysis
exclusively, the collected attached surviving cells were subjected
to FACS analysis in FACSAria Fusion (BD Biosciences), in which cells
were sorted based on the forward and side scatter (FSC/SSC) parameters
only. Individual singlet cells were collected in a 96-well Lo-Bind
plate (Eppendorf, Hamburg, Germany) containing 5 μL of 100 mM
triethylammonium bicarbonate (TEAB) per well. A total of 96 single
cells were sorted for each condition/drug (untreated and treated cells)
using separate plates. In addition, a third plate was prepared, being
dedicated only to CP, isolating 200 cells (100 treated and 100 control)
per well in the first two rows. Altogether, 24 wells of CP cells were
collected for each treatment and time point.
exclusively, the collected attached surviving cells were subjected
to FACS analysis in FACSAria Fusion (BD Biosciences), in which cells
were sorted based on the forward and side scatter (FSC/SSC) parameters
only. Individual singlet cells were collected in a 96-well Lo-Bind
plate (Eppendorf, Hamburg, Germany) containing 5 μL of 100 mM
triethylammonium bicarbonate (TEAB) per well. A total of 96 single
cells were sorted for each condition/drug (untreated and treated cells)
using separate plates. In addition, a third plate was prepared, being
dedicated only to CP, isolating 200 cells (100 treated and 100 control)
per well in the first two rows. Altogether, 24 wells of CP cells were
collected for each treatment and time point.
3
Single-cell FACS Sorting Workflow
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Collected attached cells were subjected to FACS analysis in FACSAria™ Fusion (BD Biosciences), in which cells were sorted based on the forward and side scatter (FSC/SSC) parameters only. Individual singlet cells were collected in a 96-well Lo-Bind plate (Eppendorf, Hamburg, Germany) containing 5 µL of 100 mM triethylammonium bicarbonate (TEAB) per well. A total of 96 single cells were sorted for each condition/drug (untreated and preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted September 10, 2021. ; https://doi.org/10.1101/2021.04.21.440805 doi: bioRxiv preprint treated cells) using separate plates. In addition, a third plate was prepared, being dedicated only to CP. On that plate, 200 cells (100 treated plus 100 control) were collected per well in the first two rows. Altogether, 24 wells of CP cells were collected for each treatment and time point.
The copyright holder for this this version posted September 10, 2021. ; https://doi.org/10.1101/2021.04.21.440805 doi: bioRxiv preprint treated cells) using separate plates. In addition, a third plate was prepared, being dedicated only to CP. On that plate, 200 cells (100 treated plus 100 control) were collected per well in the first two rows. Altogether, 24 wells of CP cells were collected for each treatment and time point.
4
Single-cell isolation and sorting for scRNA-seq
5
Antibody Panel Profiling for Cell Sequencing
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Antibody panels were designed to allow enrichment of cell fractions for sequencing and cell type validation. Antibodies used for FACS isolation are listed in Supplementary Table 14 . An antibody cocktail was prepared fresh by adding 3μL of each antibody in 50μL Brilliant Stain Buffer (BD) per tissue. Cells (<10x106) were resuspended in 50-100μL flow buffer and an equal volume of antibody mix was added to cells from each tissue. Cells were stained for 30 minutes on ice, washed with flow buffer and resuspended at 10x106 cells/mL. DAPI (Sigma-Aldrich) was added to a final concentration of 3μM immediately prior to sorting. Flow sorting was performed on a BD FACSAria™ Fusion instrument using DIVAv8, and data analysed using FlowJo (v10.4.1, BD). Cells were gated to exclude dead cells and doublets, and then isolated for scRNA-seq analysis (10x or Smart-seq2). For 10x, cells were sorted into chilled FACS tubes coated with FBS and prefilled with 500μL sterile PBS. For Smart-seq2, single cells were index-sorted into 96-well lo-bind plates (Eppendorf) containing 10μL lysis buffer (TCL (Qiagen) + 1% (v/v) β-mercaptoethanol) per well. B cells were also investigated by flow cytometry as per Roy et. al.54 (link).
6
Antibody Panel Profiling for Cell Sequencing
7
Single-Cell RNA-Seq Sample Preparation
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Immediately following isolation and counting, cells were collected by
centrifugation (500g for 5 min at 4°C) and resuspended in a residual
buffer. Three microliters of CD45 BUV395 (clone: HI30, BD Biosciences) was added
to the resuspended cells and incubated on ice in the dark for 30 min, washed
with Flow Buffer and resuspended at ˜1×107 cells/ml.
Immediately prior to sorting, cells were passed through a 35-µm filter
(Falcon) and DAPI (Sigma-Aldrich) was added at a final concentration of 3
μM. Flow sorting was performed on a BD FACSAria Fusion instrument using
DIVA v.8, and data were analyzed using FlowJo (v.10.4.1, BD Biosciences). Cells
were gated to exclude dead cells and doublets, and then isolated for scRNA-seq
analysis (droplet-based 10x Genomics, or plate-based Smart-seq2) using a
100-µm nozzle. For droplet-based scRNA-seq, CD45+ and
CD45− cells were sorted into separate chilled
fluorescence-activated cell sorting (FACS) tubes coated with FBS and prefilled
with 500 µl of sterile PBS. For plate-based scRNA-seq,
CD45−AF+SSC++ single cells were
index-sorted into 96-well LoBind plates (Eppendorf) containing 10 µl of
lysis buffer (TCL (Qiagen) + 1% (v/v) β-mercaptoethanol) per well.
centrifugation (500g for 5 min at 4°C) and resuspended in a residual
buffer. Three microliters of CD45 BUV395 (clone: HI30, BD Biosciences) was added
to the resuspended cells and incubated on ice in the dark for 30 min, washed
with Flow Buffer and resuspended at ˜1×107 cells/ml.
Immediately prior to sorting, cells were passed through a 35-µm filter
(Falcon) and DAPI (Sigma-Aldrich) was added at a final concentration of 3
μM. Flow sorting was performed on a BD FACSAria Fusion instrument using
DIVA v.8, and data were analyzed using FlowJo (v.10.4.1, BD Biosciences). Cells
were gated to exclude dead cells and doublets, and then isolated for scRNA-seq
analysis (droplet-based 10x Genomics, or plate-based Smart-seq2) using a
100-µm nozzle. For droplet-based scRNA-seq, CD45+ and
CD45− cells were sorted into separate chilled
fluorescence-activated cell sorting (FACS) tubes coated with FBS and prefilled
with 500 µl of sterile PBS. For plate-based scRNA-seq,
CD45−AF+SSC++ single cells were
index-sorted into 96-well LoBind plates (Eppendorf) containing 10 µl of
lysis buffer (TCL (Qiagen) + 1% (v/v) β-mercaptoethanol) per well.
8
Single-cell RNA extraction from coacervates
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RNA-containing coacervates (initial volume min. 250 µl) were sorted with a BD FACSAria Fusion flow cytometer using a 150 µl nozzle. Single-coacervates were index-sorted in precooled skirted twin.tec 96-well LoBind Plates (Eppendorf) containing 4 µl of 6 M Guanidine HCl (GuaHCl, Sigma) as lysis buffer. For each plate, one well was sorted with 1000 coacervates and one well was left empty as positive and negative controls respectively. Directly after sorting the plates were briefly spun down (max speed) to collect all FACS-derived droplets in the lysis buffer. The plates were then immediately put on dry ice until all other plates were sorted. Plates were kept at −80 °C until cDNA was prepared.
9
Single-Cell RNA-Seq Sample Preparation
10
Immunophenotyping and Sorting of Thymus Cells
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Isolated thymus cells were stained with a panel of antibodies prior to sorting based on CD45 or CD3 expression gate. The anti-human monoclonal antibodies used for flow cytometry based immunophenotyping and sorting are listed in Table S1. An antibody cocktail was freshly prepared by adding 3µL of each antibody in 50µL Brilliant Stain Buffer (BD) per tissue. Cells (≤10x10 6 ) were resuspended in 50-100µL flow buffer and an equal volume of antibody mix was added to cells from each tissue. Cells were stained for 30 minutes on ice, washed with flow buffer and resuspended at 10x10 6 cells/mL. DAPI (Sigma-Aldrich) was added to a final concentration of 3µM immediately prior to sorting. Flow sorting was performed on a BD FACSAria™ Fusion instrument using DIVA V8, and data analysed using FlowJo V10.4.1. Cells were gated to remove dead cells and doublets, and then sorted for 10X or SS2 scRNAseq analysis. For 10X droplet microfluidic analysis, cells were sorted into chilled FACS tubes coated with FBS and prefilled with 500µL sterile PBS. Paediatric samples were sorted into 50% FCS and 50% IMDM medium (supplemented with 1% glu, 1%P/S and 10% FCS). For SS2 scRNAseq analysis, single cells were index-sorted into 96-well lo-bind plates (Eppendorf) containing 10µL lysis buffer (TCL 858 (Qiagen) + 1% (v/v) E-mercaptoethanol) per well.
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