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7 protocols using edta microtainers

1

Multidimensional Immune Profiling Post-Transplant

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Blood collection began 4 weeks post-transplant through retro-orbital puncture, collected into EDTA Microtainers (BD Biosciences, San Jose, CA, USA), and stained as previously described.27 (link) Antibodies used were mouse CD45.1/CD45.2-V500 (30-F11), CD3-Pacific Blue (17A2), CD4-allophycocyanin (APC) (RM4-5), CD8-peridinin-chlorophyll-protein (PerCP) (53-6.7), CD14-phycoerythrin (PE) (rmC5-3), CD19-APC-H7 (1D3), Lineage-V450, c-Kit-APC (2B8), Sca1-PE-Cy7 (D7), CD150-PerCP/Cy5.5 (TC15-12F12.2), and CD48-APC-Cy7 (HM48-1) (BD Biosciences, BioLegend [San Diego, CA, USA], eBioscience [Waltham, MA, USA]). Lineage subsets were gated based on side scatter with CD45 on the x axis (Figure S4). Lymphocyte subsets were further analyzed based on surface markers (Figure S4). DNA was extracted (QIAGEN, Hilden, Germany) and analyzed as previously described.23 (link)
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2

Mouse Blood Sampling and Analysis

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Mouse peripheral blood was collected from submandibular vein using lancet (MEDIpoint) and blood was collected in EDTA microtainers (#365974, BD biosciences) at baseline and every two weeks post-infection. At terminal time point blood was collected via vena cava. Blood was either used to isolate plasma or to perform Flow cytometry to estimate cell counts for different leukocyte subtypes.
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3

Cross-sectional Malaria Seroprevalence Survey

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An environmentally stratified, population-based cross-sectional survey was conducted, as described by Fornace et al. 22 (link). A non-self-weighting two-stage sampling design of 919 villages was used to estimate seroprevalence to various malaria species; this analysis focuses on antibody responses to antigens from the human malaria species P. falciparum and P. vivax. The villages were stratified by forest cover, with a target sample size of 2650 households. All individuals who had been residing in selected households for the past month were asked to participate. Finger prick blood sampling was performed to prepare blood smears for the detection of malaria parasites by microscopy, and whole blood spots were collected using finger-prick blood sampling and EDTA microtainers (BD). Samples were spun to separate plasma and red blood cells and stored at − 20 °C. Participants also completed a questionnaire survey on individual and household information including demographic, health and socio-economic indicators. Location data was recorded for all households using a handheld GPS receiver (Garmin, USA).
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4

Comprehensive Blood Cell Phenotyping

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Antibodies were from Pharmingen unless otherwise specified. Peripheral blood was obtained by lancing the facial vein and collecting several drops into EDTA microtainers (Pharmingen). Blood counts were obtained using a Hemavet 50FS. Blood elements were enumerated using PE or PerCP-Cy5.5-anti-CD3 (145-2C11), PE-anti-B220 (RA3-6B2), APC-anti-CD19 (1D3), APC, PE or PerCP-Cy5.5-anti-CD11b (M1/70), PE or PerCP-Cy5.5-anti-Ter119 (Ter119), and PerCP-Cy5.5 or APC-anti-Gr-1 (RB6-8C5, Biolegend). Stem and progenitor cells were enumerated using biotin-anti-Lineage Cocktail, PerCP-Cy5.5-streptavidin, APC-anti-c-Kit (2B8) and PE-Cy7-anti-Sca-1 (D7, eBioscience), in addition to PE-anti-CD16/CD32 (FcγR, 2.4G2) and Brilliant Violet 421-anti-CD34 (RAM34) for CMP, GMP, and MEP, PE-Texas Red-anti-CD127 (IL7R, SB199) for CLP, or Brilliant Violet 421-anti-CD34 and PE-anti-CD135 (FLT3, A2F10.1) for MPP, ST-HSC, and LT-HSC. Alternatively, LSK cells were stained using PE—anti-CD150 (Q38-480) and Brilliant Violet 421–anti-CD48 (HM48-1) for LSK/SLAM LT-HSC. Marrow subsets for RNA analysis were obtained after lineage-depletion and antibody staining via a FACSAria II cell sorter (BD Biosciences).
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5

Isolation and Analysis of Immune Cells

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Peripheral blood was obtained by intra-cardiac puncture and collected in EDTA microtainers (BD Biosciences, CA, USA). Bone marrow cells were harvested by flushing single femur and tibia with ice cold PBS containing 2% fetal bovine serum (FBS, Thermo Fisher Scientific, MA, USA). Total and differential cell count was determined using a Hemavet hematology analyzer with veterinary software (Drew Scientific Inc., CT, USA).
Lamina propria cells were isolated and analyzed by flow cytometry as previously described 44 (link) and in supplementary methods. Apoptosis of cecal tissue neutrophils was enumerated using Annexin V/7AAD-apoptosis assay kit (BD biosciences, CA, USA) as per manufacturer's instructions. Flow cytometry of apoptosis assay was performed on a BD FACS Acuri™ C6 flow cytometer equipped with CFlow® Plus (BD Biosciences, CA, USA) software and analyzed using FlowJo V.10 (Tree Star, OR, USA).
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6

Isolation and Analysis of Immune Cells

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Peripheral blood was obtained by intra-cardiac puncture and collected in EDTA microtainers (BD Biosciences, CA, USA). Bone marrow cells were harvested by flushing single femur and tibia with ice cold PBS containing 2% fetal bovine serum (FBS, Thermo Fisher Scientific, MA, USA). Total and differential cell count was determined using a Hemavet hematology analyzer with veterinary software (Drew Scientific Inc., CT, USA).
Lamina propria cells were isolated and analyzed by flow cytometry as previously described 44 (link) and in supplementary methods. Apoptosis of cecal tissue neutrophils was enumerated using Annexin V/7AAD-apoptosis assay kit (BD biosciences, CA, USA) as per manufacturer's instructions. Flow cytometry of apoptosis assay was performed on a BD FACS Acuri™ C6 flow cytometer equipped with CFlow® Plus (BD Biosciences, CA, USA) software and analyzed using FlowJo V.10 (Tree Star, OR, USA).
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7

Surgical Induction of Chronic Kidney Disease

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CKD was induced by surgical 5/6th nephrectomy (SNX) as published,36 (link) with minor adaptations. In short, in a single procedure, surgical removal of the whole left kidney and polectomy (2/3rd) of the right kidney was performed using retroperitoneal incisions. In Sham animals, both kidneys were externalized and the renal capsule was removed. Postoperative buprenorphine (0.03 mg/kg Temgesic) was given subcutaneously for a period of at least 36 h. Rats were weighed 3 times a week, 24-h urines, and blood samples were collected and the SBP was measured (Tail cuff sphygmomanometer–LE 5002 LETICA®) biweekly. To collect 24-h urines, rats were placed in metabolic cages without food for 24 h, but with free access to water with 2% glucose. Urine was collected in antibiotic/antimycotic solution (A5955; Sigma, St Louis, US) and stored at −20 °C. Blood samples were collected from the tail vein into EDTA Microtainers (BD #365974). Urinary protein levels were measured by Bradford Assay (Bio Rad). Plasma urea was determined by DiaSys Urea CT FS (DiaSys Diagnostic Systems). Plasma creatinine levels were determined by DiaSys Creatinine PAP FS kit (DiaSys Diagnostic Systems,). eGFR was calculated with a plasma creatinine- and urea-based equation specific for rats.37 (link) We determined CKD as established once a threshold of proteinuria exceeding 50 mg/24 h was reached.
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