mouse anti human cd27 pe cy7  (Thermo Fisher)

Journal: International Journal of Molecular Sciences

Article Title: High Pretransplant BAFF Levels and B-cell Subset Polarized towards a Memory Phenotype as Predictive Biomarkers for Antibody-Mediated Rejection

doi: 10.3390/ijms21030779

Figure Lengend Snippet: ( a ). Gating strategy of B cell subpopulations. Lymphocytes were identified by FSC (forward scatter) and SSC (side scatter). From lymphocyte population, CD19 + cells were selected, and based on them and according to IgD and CD27 markers, naïve (IgD + CD27 - ), unswitched memory (IgD + CD27 + ), and switched memory (IgD - CD27 + ) B cells were identified. Furthermore, based on CD19 + cells and according to IgD and CD38 markers, Bm1 (IgD + CD38 - ), Bm2 (IgD + CD38 + ), Bm2’ (IgD + CD38 high ), Bm3Bm4 (IgD - CD38 high ), eBm5 (IgD - CD38 + ), and Bm5 (IgD - CD38 - ) cells were identified. Transitional type 2 cells were selected using a different gating strategy in order to obtain a pure population. For this gating strategy, CD19 + lymphocytes were selected, and from them those cells CD10 + , CD20 + , CD21 + , CD5 - , CD24 + , and CD38 + . ( b ). Gating strategy of CD4 + and CD8 + T cell subpopulations. Lymphocytes were identified by FSC (forward scatter) and SSC (side scatter). From lymphocyte population, CD3 + cells were selected, and based on them, CD4 + and CD8 + cells. Since CD4 + and CD8 + cells, and according to CD62L and CD45RO markers, naïve (CD62L + CD45RO - ), central memory (CD62L + CD45RO + ), effector memory (CD62L - CD45RO + ), and TEMRA (CD62L - CD45RO - ) T cells were identified.

Article Snippet: The following monoclonal antibodies were used to T cell subpopulations identification: anti-CD62L-FITC clone DREG56 (Beckman Coulter, Brea, CA, USA), CD45RO-PE clone UCHL1 (BD Biosciences, San Diego, CA), CD28-PC5.5 clone L293, CD27-PE Cy7 Vio770 clone 1A4CD27, CCR7-APC clone REA108 (Miltenyi Biotec, Bergisch Gladbach, Germany), CD4-APC Vio770 clone VIT4, and CD3-VioBlue clone UCHT1 (Immunostep, Salamanca, Spain).

Techniques:

Journal: Frontiers in Immunology

Article Title: Isolation and expansion of pure and functional γδ T cells

doi: 10.3389/fimmu.2024.1336870

Figure Lengend Snippet: Antibodies.

Article Snippet: Mouse anti human CD27 PE-Cy7 , Invitrogen , 25-0279-42 , 1:200.

Techniques:

Journal: Frontiers in Immunology

Article Title: Isolation and expansion of pure and functional γδ T cells

doi: 10.3389/fimmu.2024.1336870

Figure Lengend Snippet: Cell count and fold expansion of Vδ2 + T cells after 14 days of expansion. The cells were isolated through mouse anti-human Vδ2 TCR and anti-mouse IgG bead MACS enrichment, with or without additional FACS sort, and expanded straight after isolation or previously expanded Vδ2 + T cells were submitted to a new round of expansion. Expanded cells were stained for CD27 and CD45RA to determine their differentiation status. (A) Representative flow cytometry plots and gating strategy showing the percentage of Vδ2 + T cells in unsorted PBMCs, after Vδ2 TCR specific MACS isolation and after an additional FACS sort for Vδ2 + T cells. (B) The number of Vδ2 + T cells used at the start of expansion, 15.000 and 150.000, and the number of cells yielded after the 14-day expansion ( n=3 ). (C) The fold expansion of the Vδ2 + T cells for each donor of day 7, 10 and 14 relative to the start numbers, 15.000 and 150.0000, respectively ( n=3 ). (D) Flow cytometry plots showing the percentage of the Vδ2 + cells after isolation (top row) using only the anti-Vδ2 TCR antibody combined with anti-mouse IgG beads and MACS separation, without FACS sort, and after a 14-day expansion period (bottom row) for four donors. (E) The percentage of Vδ2 + T cells after 7, 10 and 14 days of expansion and (F) the percentage of CD3 + Vδ2 - T cells and CD3 + Vδ2 + T cells before and after 14 days of expansion. (G) Representative flow cytometry plots showing the frequency of CD3 + Vδ2 - and CD3 + Vδ2 + T cells in PMBCs ( left plot ) and the frequency of naïve (T naive , CD27 + CD45RA + ), terminally differentiated Effector Memory RA (T EMRA , CD27 - CD45RA + ), Effector Memory (T EM , CD27 - CD45RA - ) and Central Memory (T CM , CD27 + CD45 - ) T cells for the CD3 + Vδ2 - ( middle plot ) and CD3 + Vδ2 + T cells ( right plot ) in PMBCs before isolation. (H) Summary of the percentage of T naive , T EMRA , T EM and T CM subsets in CD3 + Vδ2 + T cells before isolation, as determined in (H, I) Flow cytometry plots of three donors showing the distribution of the T naive , T EMRA , T EM and T CM subsets in the total Vδ2 + T cell population after a 14-day expansion culture. Gating is based on freshly isolated CD3 + cells in total PMBCs from a reference donor. (J) Summary of the data in (I, K) The fold expansion of Vδ2 + T cells that were expanded for 14 days that were, straight from culture, submitted to a new expansion culture of 14-days ( n=4 ). (L) Fold expansion of Vδ2 + T cells that were previously expanded for 14 days after which they were cryopreserved for at least four weeks, thawed and submitted to a new expansion culture of 14 days ( n=4 ). The data is shown as the mean and standard deviation of the donors and data from each donor represents the mean of triplicates. Data was analyzed by a one-way ANOVA followed by Tukey’s multiple comparisons test (B, C, F, H, J, K) or a student’s T-test (L) .

Article Snippet: Mouse anti human CD27 PE-Cy7 , Invitrogen , 25-0279-42 , 1:200.

Techniques: Cell Counting, Isolation, Staining, Flow Cytometry, Standard Deviation

Journal: Cell

Article Title: In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies

doi: 10.1016/j.cell.2021.06.021

Figure Lengend Snippet: Isolation of SARS-CoV-2-reactive Abs from single cell-sorted plasmablasts and memory B cells of SARS-CoV-2 and SARS-CoV-1 convalescent donors, related to (A) Symptom severity scores of the COVID-19 convalescent donor. The method to determine severity score is in supplementary online material. Red arrows indicate the blood sampling time points that we used to isolate Abs. (B) Viral load from nasopharyngeal (NP) swabs. (C) Serum micro-neutralization titer. Micro-Neutralization titers were defined as the highest serum dilution that neutralize all the virus, or 99% inhibitory concentration (IC 99 ). (D) Flow cytometry gating strategy for unbiased plasmablasts sorting or antigen specific-memory B cells sorting. At day 11 and day 15 post onset of COVID-19 symptom, plasmablasts (CD14 - /CD16 - /CD3 - /CD235a - /CD19 + /CD20 low /IgD - /CD27 high /CD38 high ) from a SARS-CoV-2 donor. Antigen specific B cells from SARS-CoV-1 and SARS-CoV-2 donors were sorted with different combinations of the SARS-CoV-2 S-2P, RBD, NTD probes. Representative data for sorting Spike double positive, Spike + or NTD + , as well as RBD + or NTD + subsets were shown. (E-H) RBD Ab neutralization activity. (E) Proportion of SARS-CoV-2 RBD Abs (n = 81) that exhibited detectable neutralization in the microneutralization assay. (F) Neutralization IC 50 and IC 80 of RBD neutralizing Abs (NAbs) against pseudotyped SARS-CoV-2. (G) Microneutralization titer, plaque reduction neutralization test (PRNT) IC 50 and IC 80 of RBD NAbs against replication-competent SARS-CoV-2. Microneutralization titer was defined as the lowest Ab concentration that neutralized all the virus, or 99% inhibitory concentration (IC 99 ). Abs with undetectable microneutralization titers are shown as gray symbols and nAbs are represented by blue symbols. (H) RBD NAbs blocking of ACE2 binding to SARS-CoV-2 Spike (S) protein. Blocking titer is shown as IC 50 . (I-J) Correlation analysis of RBD Abs between neutralization and ACE2 blocking activities. Spearman correlation analysis were performed for (I) ACE2 blocking IC 50 versus PV neutralization IC 50 , as well as (J) for ACE2 blocking IC 50 versus SARS-CoV-2 neutralization titers (indicated by the lowest concentration that shows no CPE). Purified RBD Abs in and that have pseudovirus neutralization data (n = 59) or SARS-CoV-2 micro-neutralization assay data (n = 80) were used in this analysis. P values (p)and correlation coefficients (r) are indicated for each figure. (K-M) Neutralization activity of NTD Abs. (K) Proportion of SARS-CoV-2 NTD Abs (n = 41) that exhibited detectable neutralization in the microneutralization assay. (L) Neutralization IC 50 and IC 80 of NTD neutralizing Abs against pseudotyped SARS-CoV-2. (M) Microneutralization titer, PRNT IC 50 and IC 80 of NTD neutralizing Abs against replication-competent SARS-CoV-2. Abs with undetectable microneutralization titers are shown as gray symbols and neutralizing Abs are represented by orange symbols. Horizontal bars represent the geometric means for each group of Abs.

Article Snippet: PE-Cy7 Mouse Anti-Human CD27, Clone# O323 , eBioscience , Cat# 25-0279, RRID: AB_1724039.

Techniques: Isolation, Sampling, Neutralization, Virus, Concentration Assay, Flow Cytometry, Activity Assay, Microneutralization Assay, Plaque Reduction Neutralization Test, Blocking Assay, Binding Assay, Purification

Journal: Cell

Article Title: In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies

doi: 10.1016/j.cell.2021.06.021

Figure Lengend Snippet:

Article Snippet: PE-Cy7 Mouse Anti-Human CD27, Clone# O323 , eBioscience , Cat# 25-0279, RRID: AB_1724039.

Techniques: Virus, Clinical Proteomics, Recombinant, Staining, Reverse Transcription, Random Hexamer, Luciferase, Cell Culture, Lysis, Membrane, Multiplex Assay, Reporter Gene Assay, Binding Assay, Expressing, Transgenic Assay, Sequencing, Software