All C57BL/6 mouse studies were performed in accordance with protocols approved by the IACUC at Singapore Health Services, Singapore (ref no.: 2020/SHS/1554). C57BL/6 mice purchased from inVivos were housed in a BSL-2 animal facility at Duke-NUS Medical School. Groups of 6- to 8-week-old wild-type C57BL/6 female mice were vaccinated i.m. with either LUNAR-COV19 or conventional mRNA controls bearing the same SARS-CoV-2 S gene at doses of 0.2 μg, 2 μg, and 10 μg. For transcriptomic and T cell studies, submandibular bleeds were performed for whole blood at 24 h post-vaccination. Day 7 post-immunization, mice were sacrificed, and inguinal lymph nodes and spleens were harvested for investigation of immune gene expression and T cell responses, respectively. Splenocyte suspensions for measuring T cell responses were obtained by crushing spleen through a 70 μm cell strainer (Corning). Red blood cells were removed by lysis using BD PharmLyse reagent. For antibody studies, another set of vaccinated 6- to 8-week-old mice were bled at baseline and at 10, 20, and 30 days post-vaccination.
Pharmlyse reagent
Manufactured by BD
Sourced in United States
BD PharmLyse reagent is a laboratory product designed for the lysis of erythrocytes (red blood cells) in whole blood samples. The reagent facilitates the preparation of leukocyte (white blood cell) suspensions for subsequent analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Kaluza software, by Beckman Coulter (2 mentions)
70 μm cell strainer, by Corning (1 mentions)
Trucount tube, by BD (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Tempus spin rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Rnaeasy micro kit, by Qiagen (1 mentions)
Ncounter mouse inflammation v2 panel, by NanoString (1 mentions)
Ncounter human immunology v2 panel, by NanoString (1 mentions)
Bnt162b2, by Pfizer (1 mentions)
5 protocols using pharmlyse reagent
1
LUNAR-COV19 Vaccine Evaluation in Mice
2
Immunophenotyping of Peripheral Blood
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Peripheral blood was collected into EDTA Vacutainers. We transferred 100 µL of whole blood into Trucount™ tubes (BD Biosciences) using reverse pipetting. Cells were stained using a 10-color panel, containing fluorescently labeled monoclonal antibodies (mAbs) specific for the major immune cell populations. The OVIS panel included the following: anti-CD8 (FITC), anti-CD19 (PE), anti-CD28 (ECD), anti-CD56 (PE-Cy5), anti-CD3 (PE-Cy7), anti-CD45RA (APC), anti-CD14 (Alexa Fluor-700), anti-CD27 (APC eFluor-780), anti-CD45 (Pacific Blue), and anti-CD4 (Krome Orange) mAbs. After a 15-min incubation at room temperature, erythrocytes were lysed by BD Pharm Lyse™ reagent. Cells were run through a Gallios™ flow cytometer (Beckman Coulter), and data were analysed with the Kaluza Software (Beckman Coulter). The number of cells per microliter of whole blood was calculated as described by the manufacturer. For the Trucount method, 50 µL of mouse WB were added into Trucount tubes and processed as per the manufacturer’s protocol, except for the lysis buffer used.
3
Monocyte Subsets Characterization by Flow Cytometry
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We collected peripheral blood samples in EDTA tubes to analyze the monocytes. Monocytes were classified into three subsets based on the expression levels of CD14 and CD16. These subsets are the classical monocytes (CD14+CD16−), non-classical monocytes (CD14−CD16+), and intermediate monocytes (CD14+CD16+) [11 (link)]. Following a 15 min incubation in the dark at room temperature, erythrocytes were lysed using a BD. Pharm Lyse™ reagent according to the manufacturer’s protocol (BD Pharmingen, BD Biosystems, San Diego, CA, USA). Cells were run through a FACS Canto II flow cytometer (BD Biosystems), and at least 100,000 events were collected using FACS DIVA software (BD Biosystems). Data were studied with the Kaluza Software (Beckman Coulter, Jersey City, New Jersey), and the results are expressed using a total of monocytes (CD14+) and the percentage of each monocyte subset based on the cells’ positivity for CD14 and CD16 expression [monocytes classic (CD14+/CD16−), non-classic (CD14+/CD16++) and intermediates (CD14++/CD16+)].
4
Multivariate Analysis of Vaccine Transcriptomic Profiles
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RNA from whole blood was isolated from Tempus blood RNA tubes according to the manufacturer’s protocol (Tempus Spin RNA Isolation Kit, Thermo Fisher Scientific). Moreover, 50 ng of RNA was hybridized to reporter and capture probe sets of the nCounter Human Immunology v2 panel (Nanostring Technologies) at 65 °C for 24 hours. For in vivo experiments, 100 μL of whole blood collected 1 day postvaccination was lysed with BD PharmLyse reagent. RNA was subsequently extracted with the QIAGEN RNAeasy micro kit. 50ng of RNA was hybridized to the nCounter Mouse Inflammation v2 panel (NanoString Technologies) as previously described [28 (link)]. Unsupervised PCA performed to visualize variability between immunization routes was performed with Partek Genomics Suite Analysis v.7 software. Hierarchical clustering was performed with Seaborn’s Clustermap function in Python version 3.
5
Evaluating Humoral and T-Cell Responses to BNT162b2 in hACE2 Mice
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All animal studies were conducted in accordance with protocols approved by the IACUC at Singapore Health Services, Singapore (Ref no: 2020/SHS/1554). Female B6;SJL-Tg(K18-hACE2)2Prlmn/J (hACE2) mice were purchased from In Vivos Laboratory, Singapore. Residual reconstituted BNT162b2 that were to be disposed after daily vaccination of healthcare workers at the Singapore General Hospital were collected and stored at −80 °C until use. Vaccine used in each experiment were thawed, pooled to ensure homogeneity before being aliquoted into separate syringes for inoculation. Groups of 10- to 12-week-old wild-type hACE2 mice were vaccinated via the s.c. or i.m. route with 50 μL of BNT162b2 (Pfizer-BioNTech) for a final dosage of 5 μg. Submandibular bleeds were performed for serum isolation to assess SARS-CoV-2–specific humoral immune responses and transcriptomic studies. Ten days postprime and boost, mice were humanely killed, and spleens harvested for investigation of T-cell responses as previously reported. Briefly, spleens were passed through a 70 μm cells strainer (Corning, USA). Red blood cells were removed by lysis with BD PharmLyse reagent.
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