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CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The <t>PBMC</t> and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, <t>non-cryopreserved</t> PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.
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1) Product Images from "Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays"

Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

Journal: Cells

doi: 10.3390/cells1030409

CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The PBMC and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, non-cryopreserved PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.
Figure Legend Snippet: CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The PBMC and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, non-cryopreserved PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.

Techniques Used: Ex Vivo, Standard Deviation, Isolation, Enzyme-linked Immunospot

Comparison of mumps antigen elicited IFN-γ in freshly thawed and rested cryopreserved PBMC for low ( A ) and high responder subjects ( B ). Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or rested, the medium background was less than 10 spots per well. Non-parametric Wilcoxon signed-rank test was used to compare matched fresh vs . rested responses with a p -value ≤ 0.05 being considered significant.
Figure Legend Snippet: Comparison of mumps antigen elicited IFN-γ in freshly thawed and rested cryopreserved PBMC for low ( A ) and high responder subjects ( B ). Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or rested, the medium background was less than 10 spots per well. Non-parametric Wilcoxon signed-rank test was used to compare matched fresh vs . rested responses with a p -value ≤ 0.05 being considered significant.

Techniques Used:

Comparison of CEF responses elicited by freshly thawed (“fresh”) and rested cryopreserved PBMC in ELISPOT assays. ( A ) CEF-specific responses among “low responders” who were defined as subjects who had fewer than 50 SFU in freshly thawed PMBC. ( B ) CEF-specific responses among “high responders” who were defined as subjects who had greater than 50 spots forming units (SFU) in freshly thawed PMBC. Data points obtained from the same donor before and after resting are connected by a line. Each data point represents the mean of triplicate antigen-stimulated wells. For each donor and antigen tested, the standard deviation was
Figure Legend Snippet: Comparison of CEF responses elicited by freshly thawed (“fresh”) and rested cryopreserved PBMC in ELISPOT assays. ( A ) CEF-specific responses among “low responders” who were defined as subjects who had fewer than 50 SFU in freshly thawed PMBC. ( B ) CEF-specific responses among “high responders” who were defined as subjects who had greater than 50 spots forming units (SFU) in freshly thawed PMBC. Data points obtained from the same donor before and after resting are connected by a line. Each data point represents the mean of triplicate antigen-stimulated wells. For each donor and antigen tested, the standard deviation was

Techniques Used: Enzyme-linked Immunospot, Standard Deviation

2) Product Images from "An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi"

Article Title: An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

Journal: Cells

doi: 10.3390/cells2030607

Comparison of detection of Borrelia -specific T cells in peripheral blood by the iSpot Lyme assay and conventional ELISPOT assay. ( A ) The frequency of r Borrelia antigen-induced IFN-γ spot was established under both conditions in peripheral blood mononuclear cells (PBMC) of Borrelia positive patients. Data points obtained from the same donor with the iSpot Lyme assay and conventional ELISPOT assay are connected by a line. Each data point represents the mean spot forming unit (SFU) of triplicate antigen-stimulated wells minus the mean SFU of the corresponding medium control wells. A non-parametric Mann-Whitney U test was used to compare the matched results with a p -value of
Figure Legend Snippet: Comparison of detection of Borrelia -specific T cells in peripheral blood by the iSpot Lyme assay and conventional ELISPOT assay. ( A ) The frequency of r Borrelia antigen-induced IFN-γ spot was established under both conditions in peripheral blood mononuclear cells (PBMC) of Borrelia positive patients. Data points obtained from the same donor with the iSpot Lyme assay and conventional ELISPOT assay are connected by a line. Each data point represents the mean spot forming unit (SFU) of triplicate antigen-stimulated wells minus the mean SFU of the corresponding medium control wells. A non-parametric Mann-Whitney U test was used to compare the matched results with a p -value of

Techniques Used: Enzyme-linked Immunospot, MANN-WHITNEY

Comparison of sensitivity and specificity for detecting Borrelia infection via measuring T cell immunity by ELISPOT vs. serum antibodies by Western Blot. ( A ) Lyme ELISPOT assays and Western Blot assay were performed on PBMC and serum of 22 clinically diagnosed Borrelia patients. The percentage of individuals who scored positive for each assay is shown. ( B ) Cross-reactivity was assessed in 23 subjects with other clinical conditions, as defined in Materials and Methods, using the iSpot Lyme, conventional ELISPOT and Western Blot assay.
Figure Legend Snippet: Comparison of sensitivity and specificity for detecting Borrelia infection via measuring T cell immunity by ELISPOT vs. serum antibodies by Western Blot. ( A ) Lyme ELISPOT assays and Western Blot assay were performed on PBMC and serum of 22 clinically diagnosed Borrelia patients. The percentage of individuals who scored positive for each assay is shown. ( B ) Cross-reactivity was assessed in 23 subjects with other clinical conditions, as defined in Materials and Methods, using the iSpot Lyme, conventional ELISPOT and Western Blot assay.

Techniques Used: Infection, Enzyme-linked Immunospot, Western Blot

Optimization and validation of the iSpot Lyme assay. ( A ) Intra-assay precision. Five PBMC samples with different r Borrelia -triggered SFU response levels were tested in triplicate wells each. Bars with the specified shades show the reactivity for the three individual wells, and the mean of the three. The coefficient of variation for the replicate wells was calculated. ( B ) Inter-assay precision. Cryopreserved PBMC aliquots of the specified three Lyme patients were tested for r Borrelia reactivity on five consecutive days. The coefficient of variation for inter assay variation was calculated. ( C ) Relationship between PBMC numbers plated in each well and the IFN-γ SFU in a Borrelia positive subject, and ( D ), in a healthy control. Open symbols represent the mean of triplicate antigen-stimulated (treated) wells, the closed symbols represent the mean of the corresponding medium (control) wells. The Standard Deviation (SD) for the triplicate is smaller than the symbol when not visible. ( E ) Dose response curve for r Borrelia antigen-stimulated PBMC. ( F ) Correlation of the frequency of IFN-γ secreting Borrelia -specific T cells assessed by the iSpot Lyme assay and the concentrations of IFN-γ in the culture supernatant as measured by Bio-Plex suspension array. The nonparametric Spearman’s test was used to determine the correlation. The results showed a p -value
Figure Legend Snippet: Optimization and validation of the iSpot Lyme assay. ( A ) Intra-assay precision. Five PBMC samples with different r Borrelia -triggered SFU response levels were tested in triplicate wells each. Bars with the specified shades show the reactivity for the three individual wells, and the mean of the three. The coefficient of variation for the replicate wells was calculated. ( B ) Inter-assay precision. Cryopreserved PBMC aliquots of the specified three Lyme patients were tested for r Borrelia reactivity on five consecutive days. The coefficient of variation for inter assay variation was calculated. ( C ) Relationship between PBMC numbers plated in each well and the IFN-γ SFU in a Borrelia positive subject, and ( D ), in a healthy control. Open symbols represent the mean of triplicate antigen-stimulated (treated) wells, the closed symbols represent the mean of the corresponding medium (control) wells. The Standard Deviation (SD) for the triplicate is smaller than the symbol when not visible. ( E ) Dose response curve for r Borrelia antigen-stimulated PBMC. ( F ) Correlation of the frequency of IFN-γ secreting Borrelia -specific T cells assessed by the iSpot Lyme assay and the concentrations of IFN-γ in the culture supernatant as measured by Bio-Plex suspension array. The nonparametric Spearman’s test was used to determine the correlation. The results showed a p -value

Techniques Used: Intra Assay, Inter Assay, Standard Deviation

3) Product Images from "Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify"

Article Title: Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify

Journal: Cells

doi: 10.3390/cells7050045

Qualification of detecting HCMV-specific B cells by ImmunoSpot® testing of PBMC. ( A ) Establishing the specificity of the assay. Membranes were coated with either I-HCMV, or with anti-kappa/lambda capture antibody as a positive control, as specified. PBMC of Donor 1 of Donor 72 were added after polyclonal stimulation and plate-bound IgG was detected as described in Materials and Methods. Representative wells are shown. ( B ) Correlation between cell numbers plated and HCMV SFU detected. Preactivated PBMC of Donor 1 were plated in the specified cell numbers and the IgG SFU were counted. The result of regression analysis for the experimental data approaching linearity is specified by the R 2 value. ( C ) Identifying the IgG subclasses of HCMV-specific B cells. Wells were coated with I-HCMV antigen followed by plating of preactivated PBMC of Donor 1. The plate-bound antibodies were visualized using detection antibodies specific for pan-IgG, IgG1, IgG2, and IgG3 as specified.
Figure Legend Snippet: Qualification of detecting HCMV-specific B cells by ImmunoSpot® testing of PBMC. ( A ) Establishing the specificity of the assay. Membranes were coated with either I-HCMV, or with anti-kappa/lambda capture antibody as a positive control, as specified. PBMC of Donor 1 of Donor 72 were added after polyclonal stimulation and plate-bound IgG was detected as described in Materials and Methods. Representative wells are shown. ( B ) Correlation between cell numbers plated and HCMV SFU detected. Preactivated PBMC of Donor 1 were plated in the specified cell numbers and the IgG SFU were counted. The result of regression analysis for the experimental data approaching linearity is specified by the R 2 value. ( C ) Identifying the IgG subclasses of HCMV-specific B cells. Wells were coated with I-HCMV antigen followed by plating of preactivated PBMC of Donor 1. The plate-bound antibodies were visualized using detection antibodies specific for pan-IgG, IgG1, IgG2, and IgG3 as specified.

Techniques Used: Positive Control

4) Product Images from "Analysis of cellular and humoral immune responses against cytomegalovirus in patients with autoimmune Addison’s disease"

Article Title: Analysis of cellular and humoral immune responses against cytomegalovirus in patients with autoimmune Addison’s disease

Journal: Journal of Translational Medicine

doi: 10.1186/s12967-016-0822-z

Ex vivo and in vitro stimulated (IVS) frequencies of CMV specific CD8 + T cells in AAD patients and controls. Immudex CMV dextramers were used to stain PBMC directly (ex vivo), or after the cells were stimulated with HLA-restricted CMV peptide (1 µg/mL) and expanded for 13 days (in vitro stimulated), for HLA-A2 or HLA-B8 CMV specific CD8 + T cells. All samples were analyzed on an Accuri C6 flow cytometer. Results are expressed as percentage of CD8 + T cells staining positive for CMV dextramers in a patients and controls. The same data are also shown grouped into HLA-B8 ( b ) and HLA-A2 ( c ) positive patients ( b n = ex v. 9, IVS 6, c n = ex v. 6, IVS 4) and controls ( b n = ex v. 10, IVS 9, c n = ex v. 6, IVS 4). Non-parametric Mann–Whitney U test was used to test for statistical differences between patients and controls, but none were found. The bars display the mean for the whole group
Figure Legend Snippet: Ex vivo and in vitro stimulated (IVS) frequencies of CMV specific CD8 + T cells in AAD patients and controls. Immudex CMV dextramers were used to stain PBMC directly (ex vivo), or after the cells were stimulated with HLA-restricted CMV peptide (1 µg/mL) and expanded for 13 days (in vitro stimulated), for HLA-A2 or HLA-B8 CMV specific CD8 + T cells. All samples were analyzed on an Accuri C6 flow cytometer. Results are expressed as percentage of CD8 + T cells staining positive for CMV dextramers in a patients and controls. The same data are also shown grouped into HLA-B8 ( b ) and HLA-A2 ( c ) positive patients ( b n = ex v. 9, IVS 6, c n = ex v. 6, IVS 4) and controls ( b n = ex v. 10, IVS 9, c n = ex v. 6, IVS 4). Non-parametric Mann–Whitney U test was used to test for statistical differences between patients and controls, but none were found. The bars display the mean for the whole group

Techniques Used: Ex Vivo, In Vitro, Staining, Flow Cytometry, Cytometry, MANN-WHITNEY

Levels of degranulating CMV-specific CD8 + T cells in AAD patient and control cells. PBMC from AAD patients and healthy controls were stimulated with HLA-specific CMV peptide (1 µg/mL) and expanded for 13 days, before being re-stimulated with CMV peptide and stained with anti-human CD107a for 4 h. The cells were also stained with anti-human CD8 before being analyzed on an Accuri C6 flow cytometer. Results are expressed as percentage of CD8 + T cells positive for CD107a in patients and controls ( a ). The same data are also shown grouped into HLA-B8 ( b ) and HLA-A2 ( c ) positive patients ( b n = 3, c n = 4) and controls ( b n = 7, c n = 3). Non-parametric Mann–Whitney U test was used to test for statistical differences between patients and controls, but none were found. The bars display the mean for the whole group
Figure Legend Snippet: Levels of degranulating CMV-specific CD8 + T cells in AAD patient and control cells. PBMC from AAD patients and healthy controls were stimulated with HLA-specific CMV peptide (1 µg/mL) and expanded for 13 days, before being re-stimulated with CMV peptide and stained with anti-human CD107a for 4 h. The cells were also stained with anti-human CD8 before being analyzed on an Accuri C6 flow cytometer. Results are expressed as percentage of CD8 + T cells positive for CD107a in patients and controls ( a ). The same data are also shown grouped into HLA-B8 ( b ) and HLA-A2 ( c ) positive patients ( b n = 3, c n = 4) and controls ( b n = 7, c n = 3). Non-parametric Mann–Whitney U test was used to test for statistical differences between patients and controls, but none were found. The bars display the mean for the whole group

Techniques Used: Staining, Flow Cytometry, Cytometry, MANN-WHITNEY

Ex vivo and in vitro stimulated (IVS) levels of CMV-specific IFN-γ producing T cells in AAD patients and controls. IFN-γ ELISpot was used to determine the amount of CMV-specific T cells in PBMC from HLA-A2 or HLA-B8 patients or healthy controls stimulated with HLA-restricted CMV peptide (1 µg/mL) for 24 h (ex vivo) or expanded for 13 days, and then then re-stimulated with CMV peptide for 24 h (in vitro stimulated). The results are displayed as means of triplicates of spot forming units (SFUs) per 1 × 10 6 PBMCs ( a ). The same data are also shown grouped into HLA-B8 ( b ) and HLA-A2 ( c ) positive patients ( b n = ex v. 9, IVS 8, c n = ex v. 6, IVS 4) and controls ( b n = ex v. 10, IVS 10, c n = ex v. 6, IVS 4). Non-parametric Mann–Whitney U test was used to test for statistical differences between patients and controls, but none were found. The bars display the mean for the whole group
Figure Legend Snippet: Ex vivo and in vitro stimulated (IVS) levels of CMV-specific IFN-γ producing T cells in AAD patients and controls. IFN-γ ELISpot was used to determine the amount of CMV-specific T cells in PBMC from HLA-A2 or HLA-B8 patients or healthy controls stimulated with HLA-restricted CMV peptide (1 µg/mL) for 24 h (ex vivo) or expanded for 13 days, and then then re-stimulated with CMV peptide for 24 h (in vitro stimulated). The results are displayed as means of triplicates of spot forming units (SFUs) per 1 × 10 6 PBMCs ( a ). The same data are also shown grouped into HLA-B8 ( b ) and HLA-A2 ( c ) positive patients ( b n = ex v. 9, IVS 8, c n = ex v. 6, IVS 4) and controls ( b n = ex v. 10, IVS 10, c n = ex v. 6, IVS 4). Non-parametric Mann–Whitney U test was used to test for statistical differences between patients and controls, but none were found. The bars display the mean for the whole group

Techniques Used: Ex Vivo, In Vitro, Enzyme-linked Immunospot, MANN-WHITNEY

5) Product Images from "Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify"

Article Title: Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify

Journal: Cells

doi: 10.3390/cells7050045

Qualification of HCMV-specific T-cell IFN-γ ImmunoSpot® testing of PBMC. ( A ) Establishing the CD4+/CD8+ lineage of HCMV-specific memory T cells. CD4+ or CD8+ T-cell depleted PBMC or unseparated PBMC of Donor 1 were tested in an IFN-γ ImmunoSpot® assay. The numbers of I-HCMV or HCMVpp65-induced IFN-γ SFU were measured for each condition, and the mean and SD for three replicate measurements of each are shown. ( B ) PBMC cell-number dependence of SFU. PBMC of Donor 1 were plated in the specified cell numbers per well and I-HCMV or HCMVpp65 (specified by color) was added at 50 μg/mL or 1 μg/mL, respectively. The numbers of IFN-γ SFU were established. Means and SD are shown for triplicate wells. The result of regression analysis for the experimental data approaching linearity is specified for both conditions as the R 2 value in color. ( C ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by HCMVpp65 peptides. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different peptide concentrations specified. The number of IFN-γ SFU was measured in single wells. ( D ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by I-HCMV. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different antigen concentrations specified. The number of IFN-γ SFU was measured in single wells.
Figure Legend Snippet: Qualification of HCMV-specific T-cell IFN-γ ImmunoSpot® testing of PBMC. ( A ) Establishing the CD4+/CD8+ lineage of HCMV-specific memory T cells. CD4+ or CD8+ T-cell depleted PBMC or unseparated PBMC of Donor 1 were tested in an IFN-γ ImmunoSpot® assay. The numbers of I-HCMV or HCMVpp65-induced IFN-γ SFU were measured for each condition, and the mean and SD for three replicate measurements of each are shown. ( B ) PBMC cell-number dependence of SFU. PBMC of Donor 1 were plated in the specified cell numbers per well and I-HCMV or HCMVpp65 (specified by color) was added at 50 μg/mL or 1 μg/mL, respectively. The numbers of IFN-γ SFU were established. Means and SD are shown for triplicate wells. The result of regression analysis for the experimental data approaching linearity is specified for both conditions as the R 2 value in color. ( C ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by HCMVpp65 peptides. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different peptide concentrations specified. The number of IFN-γ SFU was measured in single wells. ( D ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by I-HCMV. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different antigen concentrations specified. The number of IFN-γ SFU was measured in single wells.

Techniques Used:

Establishing the specificity of I-HCMV- and HCMVpp65-induced IFN-γ ImmunoSpot® formation. Donor 1 was seropositive for HCMV, Donor 72 was seronegative. PBMC from both donors were tested in an IFN-γ ImmunoSpot® assay in medium alone as the negative control (“MEDIUM”), or in the presence of the HCMVpp65 peptide pool (HCMVpp65) or inactivated HCMV virions (I-HCMV), as specified. As positive controls for eliciting memory T-cell responses, a pool of 32 peptides of HCMV, EBV, and influenza virus were used (CEFpp). The test was performed as specified in Materials and Methods, with three replicate wells for each condition. A representative image of one of the replicates is shown for each condition.
Figure Legend Snippet: Establishing the specificity of I-HCMV- and HCMVpp65-induced IFN-γ ImmunoSpot® formation. Donor 1 was seropositive for HCMV, Donor 72 was seronegative. PBMC from both donors were tested in an IFN-γ ImmunoSpot® assay in medium alone as the negative control (“MEDIUM”), or in the presence of the HCMVpp65 peptide pool (HCMVpp65) or inactivated HCMV virions (I-HCMV), as specified. As positive controls for eliciting memory T-cell responses, a pool of 32 peptides of HCMV, EBV, and influenza virus were used (CEFpp). The test was performed as specified in Materials and Methods, with three replicate wells for each condition. A representative image of one of the replicates is shown for each condition.

Techniques Used: Negative Control

Kinetics of I-HCMV-induced IFN-γ production. Donors 2 and 4 were seronegative for HCMV but were shown to respond to I-HCMV in previous experiments. Donor 56 was also HCMV-seronegative and was shown not to respond to I-HCMV (see Table 1 ). Donors 84, 85, and 86 were HCMV seropositive. The PBMC of all six donors were retested in an IFN-γ ImmunoSpot® assay at 3 × 10 5 cells per well after 6, 12, 24, and 48 h antigen stimulation periods with 50 μL/mL of I-HCMV. Each condition was tested in triplicate wells, of which one representative well is shown.
Figure Legend Snippet: Kinetics of I-HCMV-induced IFN-γ production. Donors 2 and 4 were seronegative for HCMV but were shown to respond to I-HCMV in previous experiments. Donor 56 was also HCMV-seronegative and was shown not to respond to I-HCMV (see Table 1 ). Donors 84, 85, and 86 were HCMV seropositive. The PBMC of all six donors were retested in an IFN-γ ImmunoSpot® assay at 3 × 10 5 cells per well after 6, 12, 24, and 48 h antigen stimulation periods with 50 μL/mL of I-HCMV. Each condition was tested in triplicate wells, of which one representative well is shown.

Techniques Used:

Qualification of detecting HCMV-specific B cells by ImmunoSpot® testing of PBMC. ( A ) Establishing the specificity of the assay. Membranes were coated with either I-HCMV, or with anti-kappa/lambda capture antibody as a positive control, as specified. PBMC of Donor 1 of Donor 72 were added after polyclonal stimulation and plate-bound IgG was detected as described in Materials and Methods. Representative wells are shown. ( B ) Correlation between cell numbers plated and HCMV SFU detected. Preactivated PBMC of Donor 1 were plated in the specified cell numbers and the IgG SFU were counted. The result of regression analysis for the experimental data approaching linearity is specified by the R 2 value. ( C ) Identifying the IgG subclasses of HCMV-specific B cells. Wells were coated with I-HCMV antigen followed by plating of preactivated PBMC of Donor 1. The plate-bound antibodies were visualized using detection antibodies specific for pan-IgG, IgG1, IgG2, and IgG3 as specified.
Figure Legend Snippet: Qualification of detecting HCMV-specific B cells by ImmunoSpot® testing of PBMC. ( A ) Establishing the specificity of the assay. Membranes were coated with either I-HCMV, or with anti-kappa/lambda capture antibody as a positive control, as specified. PBMC of Donor 1 of Donor 72 were added after polyclonal stimulation and plate-bound IgG was detected as described in Materials and Methods. Representative wells are shown. ( B ) Correlation between cell numbers plated and HCMV SFU detected. Preactivated PBMC of Donor 1 were plated in the specified cell numbers and the IgG SFU were counted. The result of regression analysis for the experimental data approaching linearity is specified by the R 2 value. ( C ) Identifying the IgG subclasses of HCMV-specific B cells. Wells were coated with I-HCMV antigen followed by plating of preactivated PBMC of Donor 1. The plate-bound antibodies were visualized using detection antibodies specific for pan-IgG, IgG1, IgG2, and IgG3 as specified.

Techniques Used: Positive Control

6) Product Images from "Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify"

Article Title: Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify

Journal: Cells

doi: 10.3390/cells7050045

Qualification of HCMV-specific T-cell IFN-γ ImmunoSpot® testing of PBMC. ( A ) Establishing the CD4+/CD8+ lineage of HCMV-specific memory T cells. CD4+ or CD8+ T-cell depleted PBMC or unseparated PBMC of Donor 1 were tested in an IFN-γ ImmunoSpot® assay. The numbers of I-HCMV or HCMVpp65-induced IFN-γ SFU were measured for each condition, and the mean and SD for three replicate measurements of each are shown. ( B ) PBMC cell-number dependence of SFU. PBMC of Donor 1 were plated in the specified cell numbers per well and I-HCMV or HCMVpp65 (specified by color) was added at 50 μg/mL or 1 μg/mL, respectively. The numbers of IFN-γ SFU were established. Means and SD are shown for triplicate wells. The result of regression analysis for the experimental data approaching linearity is specified for both conditions as the R 2 value in color. ( C ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by HCMVpp65 peptides. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different peptide concentrations specified. The number of IFN-γ SFU was measured in single wells. ( D ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by I-HCMV. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different antigen concentrations specified. The number of IFN-γ SFU was measured in single wells.
Figure Legend Snippet: Qualification of HCMV-specific T-cell IFN-γ ImmunoSpot® testing of PBMC. ( A ) Establishing the CD4+/CD8+ lineage of HCMV-specific memory T cells. CD4+ or CD8+ T-cell depleted PBMC or unseparated PBMC of Donor 1 were tested in an IFN-γ ImmunoSpot® assay. The numbers of I-HCMV or HCMVpp65-induced IFN-γ SFU were measured for each condition, and the mean and SD for three replicate measurements of each are shown. ( B ) PBMC cell-number dependence of SFU. PBMC of Donor 1 were plated in the specified cell numbers per well and I-HCMV or HCMVpp65 (specified by color) was added at 50 μg/mL or 1 μg/mL, respectively. The numbers of IFN-γ SFU were established. Means and SD are shown for triplicate wells. The result of regression analysis for the experimental data approaching linearity is specified for both conditions as the R 2 value in color. ( C ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by HCMVpp65 peptides. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different peptide concentrations specified. The number of IFN-γ SFU was measured in single wells. ( D ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by I-HCMV. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different antigen concentrations specified. The number of IFN-γ SFU was measured in single wells.

Techniques Used:

Establishing the specificity of I-HCMV- and HCMVpp65-induced IFN-γ ImmunoSpot® formation. Donor 1 was seropositive for HCMV, Donor 72 was seronegative. PBMC from both donors were tested in an IFN-γ ImmunoSpot® assay in medium alone as the negative control (“MEDIUM”), or in the presence of the HCMVpp65 peptide pool (HCMVpp65) or inactivated HCMV virions (I-HCMV), as specified. As positive controls for eliciting memory T-cell responses, a pool of 32 peptides of HCMV, EBV, and influenza virus were used (CEFpp). The test was performed as specified in Materials and Methods, with three replicate wells for each condition. A representative image of one of the replicates is shown for each condition.
Figure Legend Snippet: Establishing the specificity of I-HCMV- and HCMVpp65-induced IFN-γ ImmunoSpot® formation. Donor 1 was seropositive for HCMV, Donor 72 was seronegative. PBMC from both donors were tested in an IFN-γ ImmunoSpot® assay in medium alone as the negative control (“MEDIUM”), or in the presence of the HCMVpp65 peptide pool (HCMVpp65) or inactivated HCMV virions (I-HCMV), as specified. As positive controls for eliciting memory T-cell responses, a pool of 32 peptides of HCMV, EBV, and influenza virus were used (CEFpp). The test was performed as specified in Materials and Methods, with three replicate wells for each condition. A representative image of one of the replicates is shown for each condition.

Techniques Used: Negative Control

Qualification of detecting HCMV-specific B cells by ImmunoSpot® testing of PBMC. ( A ) Establishing the specificity of the assay. Membranes were coated with either I-HCMV, or with anti-kappa/lambda capture antibody as a positive control, as specified. PBMC of Donor 1 of Donor 72 were added after polyclonal stimulation and plate-bound IgG was detected as described in Materials and Methods. Representative wells are shown. ( B ) Correlation between cell numbers plated and HCMV SFU detected. Preactivated PBMC of Donor 1 were plated in the specified cell numbers and the IgG SFU were counted. The result of regression analysis for the experimental data approaching linearity is specified by the R 2 value. ( C ) Identifying the IgG subclasses of HCMV-specific B cells. Wells were coated with I-HCMV antigen followed by plating of preactivated PBMC of Donor 1. The plate-bound antibodies were visualized using detection antibodies specific for pan-IgG, IgG1, IgG2, and IgG3 as specified.
Figure Legend Snippet: Qualification of detecting HCMV-specific B cells by ImmunoSpot® testing of PBMC. ( A ) Establishing the specificity of the assay. Membranes were coated with either I-HCMV, or with anti-kappa/lambda capture antibody as a positive control, as specified. PBMC of Donor 1 of Donor 72 were added after polyclonal stimulation and plate-bound IgG was detected as described in Materials and Methods. Representative wells are shown. ( B ) Correlation between cell numbers plated and HCMV SFU detected. Preactivated PBMC of Donor 1 were plated in the specified cell numbers and the IgG SFU were counted. The result of regression analysis for the experimental data approaching linearity is specified by the R 2 value. ( C ) Identifying the IgG subclasses of HCMV-specific B cells. Wells were coated with I-HCMV antigen followed by plating of preactivated PBMC of Donor 1. The plate-bound antibodies were visualized using detection antibodies specific for pan-IgG, IgG1, IgG2, and IgG3 as specified.

Techniques Used: Positive Control

7) Product Images from "An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi"

Article Title: An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

Journal: Cells

doi: 10.3390/cells2030607

Comparison of detection of Borrelia -specific T cells in peripheral blood by the iSpot Lyme assay and conventional ELISPOT assay. ( A ) The frequency of r Borrelia antigen-induced IFN-γ spot was established under both conditions in peripheral blood mononuclear cells (PBMC) of Borrelia positive patients. Data points obtained from the same donor with the iSpot Lyme assay and conventional ELISPOT assay are connected by a line. Each data point represents the mean spot forming unit (SFU) of triplicate antigen-stimulated wells minus the mean SFU of the corresponding medium control wells. A non-parametric Mann-Whitney U test was used to compare the matched results with a p -value of
Figure Legend Snippet: Comparison of detection of Borrelia -specific T cells in peripheral blood by the iSpot Lyme assay and conventional ELISPOT assay. ( A ) The frequency of r Borrelia antigen-induced IFN-γ spot was established under both conditions in peripheral blood mononuclear cells (PBMC) of Borrelia positive patients. Data points obtained from the same donor with the iSpot Lyme assay and conventional ELISPOT assay are connected by a line. Each data point represents the mean spot forming unit (SFU) of triplicate antigen-stimulated wells minus the mean SFU of the corresponding medium control wells. A non-parametric Mann-Whitney U test was used to compare the matched results with a p -value of

Techniques Used: Enzyme-linked Immunospot, MANN-WHITNEY

Comparison of sensitivity and specificity for detecting Borrelia infection via measuring T cell immunity by ELISPOT vs. serum antibodies by Western Blot. ( A ) Lyme ELISPOT assays and Western Blot assay were performed on PBMC and serum of 22 clinically diagnosed Borrelia patients. The percentage of individuals who scored positive for each assay is shown. ( B ) Cross-reactivity was assessed in 23 subjects with other clinical conditions, as defined in Materials and Methods, using the iSpot Lyme, conventional ELISPOT and Western Blot assay.
Figure Legend Snippet: Comparison of sensitivity and specificity for detecting Borrelia infection via measuring T cell immunity by ELISPOT vs. serum antibodies by Western Blot. ( A ) Lyme ELISPOT assays and Western Blot assay were performed on PBMC and serum of 22 clinically diagnosed Borrelia patients. The percentage of individuals who scored positive for each assay is shown. ( B ) Cross-reactivity was assessed in 23 subjects with other clinical conditions, as defined in Materials and Methods, using the iSpot Lyme, conventional ELISPOT and Western Blot assay.

Techniques Used: Infection, Enzyme-linked Immunospot, Western Blot

Optimization and validation of the iSpot Lyme assay. ( A ) Intra-assay precision. Five PBMC samples with different r Borrelia -triggered SFU response levels were tested in triplicate wells each. Bars with the specified shades show the reactivity for the three individual wells, and the mean of the three. The coefficient of variation for the replicate wells was calculated. ( B ) Inter-assay precision. Cryopreserved PBMC aliquots of the specified three Lyme patients were tested for r Borrelia reactivity on five consecutive days. The coefficient of variation for inter assay variation was calculated. ( C ) Relationship between PBMC numbers plated in each well and the IFN-γ SFU in a Borrelia positive subject, and ( D ), in a healthy control. Open symbols represent the mean of triplicate antigen-stimulated (treated) wells, the closed symbols represent the mean of the corresponding medium (control) wells. The Standard Deviation (SD) for the triplicate is smaller than the symbol when not visible. ( E ) Dose response curve for r Borrelia antigen-stimulated PBMC. ( F ) Correlation of the frequency of IFN-γ secreting Borrelia -specific T cells assessed by the iSpot Lyme assay and the concentrations of IFN-γ in the culture supernatant as measured by Bio-Plex suspension array. The nonparametric Spearman’s test was used to determine the correlation. The results showed a p -value
Figure Legend Snippet: Optimization and validation of the iSpot Lyme assay. ( A ) Intra-assay precision. Five PBMC samples with different r Borrelia -triggered SFU response levels were tested in triplicate wells each. Bars with the specified shades show the reactivity for the three individual wells, and the mean of the three. The coefficient of variation for the replicate wells was calculated. ( B ) Inter-assay precision. Cryopreserved PBMC aliquots of the specified three Lyme patients were tested for r Borrelia reactivity on five consecutive days. The coefficient of variation for inter assay variation was calculated. ( C ) Relationship between PBMC numbers plated in each well and the IFN-γ SFU in a Borrelia positive subject, and ( D ), in a healthy control. Open symbols represent the mean of triplicate antigen-stimulated (treated) wells, the closed symbols represent the mean of the corresponding medium (control) wells. The Standard Deviation (SD) for the triplicate is smaller than the symbol when not visible. ( E ) Dose response curve for r Borrelia antigen-stimulated PBMC. ( F ) Correlation of the frequency of IFN-γ secreting Borrelia -specific T cells assessed by the iSpot Lyme assay and the concentrations of IFN-γ in the culture supernatant as measured by Bio-Plex suspension array. The nonparametric Spearman’s test was used to determine the correlation. The results showed a p -value

Techniques Used: Intra Assay, Inter Assay, Standard Deviation

8) Product Images from "Nuclease-resistant immunostimulatory phosphodiester CpG oligodeoxynucleotides as human Toll-like receptor 9 agonists"

Article Title: Nuclease-resistant immunostimulatory phosphodiester CpG oligodeoxynucleotides as human Toll-like receptor 9 agonists

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-11-88

TLR9 activation by B lymphocytes, PBMC, and CAL-1 cells . (A) Activation of NF-κB in human B lymphocyte cell lines, Ramos-Blue cells, stably transfected with NF-kB/AP-1-inducible SEAP reporter gene. n = 3, mean (sd). All P values are shown relative to the PD-ODN2006: * P
Figure Legend Snippet: TLR9 activation by B lymphocytes, PBMC, and CAL-1 cells . (A) Activation of NF-κB in human B lymphocyte cell lines, Ramos-Blue cells, stably transfected with NF-kB/AP-1-inducible SEAP reporter gene. n = 3, mean (sd). All P values are shown relative to the PD-ODN2006: * P

Techniques Used: Activation Assay, Stable Transfection, Transfection

9) Product Images from "Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify"

Article Title: Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify

Journal: Cells

doi: 10.3390/cells7050045

Qualification of detecting HCMV-specific B cells by ImmunoSpot® testing of PBMC. ( A ) Establishing the specificity of the assay. Membranes were coated with either I-HCMV, or with anti-kappa/lambda capture antibody as a positive control, as specified. PBMC of Donor 1 of Donor 72 were added after polyclonal stimulation and plate-bound IgG was detected as described in Materials and Methods. Representative wells are shown. ( B ) Correlation between cell numbers plated and HCMV SFU detected. Preactivated PBMC of Donor 1 were plated in the specified cell numbers and the IgG SFU were counted. The result of regression analysis for the experimental data approaching linearity is specified by the R 2 value. ( C ) Identifying the IgG subclasses of HCMV-specific B cells. Wells were coated with I-HCMV antigen followed by plating of preactivated PBMC of Donor 1. The plate-bound antibodies were visualized using detection antibodies specific for pan-IgG, IgG1, IgG2, and IgG3 as specified.
Figure Legend Snippet: Qualification of detecting HCMV-specific B cells by ImmunoSpot® testing of PBMC. ( A ) Establishing the specificity of the assay. Membranes were coated with either I-HCMV, or with anti-kappa/lambda capture antibody as a positive control, as specified. PBMC of Donor 1 of Donor 72 were added after polyclonal stimulation and plate-bound IgG was detected as described in Materials and Methods. Representative wells are shown. ( B ) Correlation between cell numbers plated and HCMV SFU detected. Preactivated PBMC of Donor 1 were plated in the specified cell numbers and the IgG SFU were counted. The result of regression analysis for the experimental data approaching linearity is specified by the R 2 value. ( C ) Identifying the IgG subclasses of HCMV-specific B cells. Wells were coated with I-HCMV antigen followed by plating of preactivated PBMC of Donor 1. The plate-bound antibodies were visualized using detection antibodies specific for pan-IgG, IgG1, IgG2, and IgG3 as specified.

Techniques Used: Positive Control

10) Product Images from "Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies "

Article Title: Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies

Journal: Cells

doi: 10.3390/cells1030313

Viability of cryopreserved peripheral blood mononuclear cells (PBMC) from seven subjects following thawing under warm or cold processing conditions. Warm wash medium was added to warm cells, and cold wash medium to cold cells, at a rate of 1 mL/5 seconds (slow) or in a single stream
Figure Legend Snippet: Viability of cryopreserved peripheral blood mononuclear cells (PBMC) from seven subjects following thawing under warm or cold processing conditions. Warm wash medium was added to warm cells, and cold wash medium to cold cells, at a rate of 1 mL/5 seconds (slow) or in a single stream

Techniques Used:

Effect of post-thaw DMSO exposure at 37 °C on the viability and functionality of cryopreserved PBMC. Cryovials were kept in a bead bath at 37 °C for 10 minutes, followed by an additional 0, 30, and 60 minutes as specified on the X axis. Viability ( A ), CD8 cell function ( B ) and CD4 cell function ( C ) was assessed as specified in the Legend to Figure 2 .
Figure Legend Snippet: Effect of post-thaw DMSO exposure at 37 °C on the viability and functionality of cryopreserved PBMC. Cryovials were kept in a bead bath at 37 °C for 10 minutes, followed by an additional 0, 30, and 60 minutes as specified on the X axis. Viability ( A ), CD8 cell function ( B ) and CD4 cell function ( C ) was assessed as specified in the Legend to Figure 2 .

Techniques Used: Cell Function Assay

Viability and functionality of cryopreserved PBMC from 10 subjects following thawing under warm or cold processing conditions. ( A ) Post-thaw viability of PBMC under both conditions was assessed as described in Figure 1 . ( B ) CD8 ELISPOT responses were measured using the CEF peptide pool that comprised of defined Class I peptides from CMV, EBV, and influenza viruses [ 18 , 19 ] as the antigen to stimulate the PBMC that were thawed according to the “warm-” or “cold processing protocol. ( C ). CD4 ELISPOT responses: mumps and mosquito antigen were used to stimulate CD4 cells. Statistical comparisons were performedwith Wilcoxon signed rank test with two-tailed p values ≤0.05 being considered significant.
Figure Legend Snippet: Viability and functionality of cryopreserved PBMC from 10 subjects following thawing under warm or cold processing conditions. ( A ) Post-thaw viability of PBMC under both conditions was assessed as described in Figure 1 . ( B ) CD8 ELISPOT responses were measured using the CEF peptide pool that comprised of defined Class I peptides from CMV, EBV, and influenza viruses [ 18 , 19 ] as the antigen to stimulate the PBMC that were thawed according to the “warm-” or “cold processing protocol. ( C ). CD4 ELISPOT responses: mumps and mosquito antigen were used to stimulate CD4 cells. Statistical comparisons were performedwith Wilcoxon signed rank test with two-tailed p values ≤0.05 being considered significant.

Techniques Used: Enzyme-linked Immunospot, Two Tailed Test

11) Product Images from "B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody"

Article Title: B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody

Journal: Cells

doi: 10.3390/cells7060050

Calculating frequencies for pan IgG-SFU/million PBMC illustrated. Polyclonally stimulated PBMC were plated in the specified cell numbers in quadruplicate wells, and a Three Color ImmunoSpot ® assay was performed for Ig classes pan-IgG, M and A. ( A ) Raw images for the IgG color plane are shown with the cell numbers plated per well specified; ( B ) the mean spot count for each cell concentration, with the SD for the quadruplicate wells tested is plotted vs. the cell numbers plated. As the spots at high ASC densities start crowding (plus when the number of ASC is high, there is an “ELISA” effect with analyte captured from the supernatant causing a high background carpet staining), for high cell numbers Spot Forming Units (SFU) become no longer precisely countable: “too numerous to count is shown by a star; ( C ) While at high numbers spot counts tend to deviate from linearity due to crowding, in lower numbers they followed a liner relationship between cell numbers plated and SFU counted, in this case with an R 2 value of 0.9939. Based on the regression line shown in red, the pan-IgG SFU frequency has been calculated to be, in the example shown here, 13,800 pan-IgG SFU per one million PBMC.
Figure Legend Snippet: Calculating frequencies for pan IgG-SFU/million PBMC illustrated. Polyclonally stimulated PBMC were plated in the specified cell numbers in quadruplicate wells, and a Three Color ImmunoSpot ® assay was performed for Ig classes pan-IgG, M and A. ( A ) Raw images for the IgG color plane are shown with the cell numbers plated per well specified; ( B ) the mean spot count for each cell concentration, with the SD for the quadruplicate wells tested is plotted vs. the cell numbers plated. As the spots at high ASC densities start crowding (plus when the number of ASC is high, there is an “ELISA” effect with analyte captured from the supernatant causing a high background carpet staining), for high cell numbers Spot Forming Units (SFU) become no longer precisely countable: “too numerous to count is shown by a star; ( C ) While at high numbers spot counts tend to deviate from linearity due to crowding, in lower numbers they followed a liner relationship between cell numbers plated and SFU counted, in this case with an R 2 value of 0.9939. Based on the regression line shown in red, the pan-IgG SFU frequency has been calculated to be, in the example shown here, 13,800 pan-IgG SFU per one million PBMC.

Techniques Used: Concentration Assay, Staining

12) Product Images from "Allogeneic partially HLA-matched dendritic cells pulsed with autologous tumor cell lysate as a vaccine in metastatic renal cell cancer"

Article Title: Allogeneic partially HLA-matched dendritic cells pulsed with autologous tumor cell lysate as a vaccine in metastatic renal cell cancer

Journal: Human Vaccines & Immunotherapeutics

doi: 10.4161/hv.24149

Figure 3. IFN- γ ELISpot in patients no. 3 and 7. PBMC from patients before vaccination and at different time points after vaccination were stimulated in vitro with different tumor-associated peptides from survivin, vimentin, ADFP, TYMS,
Figure Legend Snippet: Figure 3. IFN- γ ELISpot in patients no. 3 and 7. PBMC from patients before vaccination and at different time points after vaccination were stimulated in vitro with different tumor-associated peptides from survivin, vimentin, ADFP, TYMS,

Techniques Used: Enzyme-linked Immunospot, In Vitro

13) Product Images from "Identification and translational validation of novel mammaglobin-A CD8 T cell epitopes"

Article Title: Identification and translational validation of novel mammaglobin-A CD8 T cell epitopes

Journal: Breast cancer research and treatment

doi: 10.1007/s10549-014-3129-x

CD8 T cells specific for novel MAM-A epitopes recognize and lyse breast cancer cells. a HLA-A2 + , MAM-A + breast cancer cells (AU565), but not HLA-A2 + , MAM-A − breast cancer cells (MCF-7) are recognized by peptide-specific CD8 T cells. PBMC from
Figure Legend Snippet: CD8 T cells specific for novel MAM-A epitopes recognize and lyse breast cancer cells. a HLA-A2 + , MAM-A + breast cancer cells (AU565), but not HLA-A2 + , MAM-A − breast cancer cells (MCF-7) are recognized by peptide-specific CD8 T cells. PBMC from

Techniques Used:

14) Product Images from "B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody"

Article Title: B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody

Journal: Cells

doi: 10.3390/cells7060050

Calculating frequencies for pan IgG-SFU/million PBMC illustrated. Polyclonally stimulated PBMC were plated in the specified cell numbers in quadruplicate wells, and a Three Color ImmunoSpot ® assay was performed for Ig classes pan-IgG, M and A. ( A ) Raw images for the IgG color plane are shown with the cell numbers plated per well specified; ( B ) the mean spot count for each cell concentration, with the SD for the quadruplicate wells tested is plotted vs. the cell numbers plated. As the spots at high ASC densities start crowding (plus when the number of ASC is high, there is an “ELISA” effect with analyte captured from the supernatant causing a high background carpet staining), for high cell numbers Spot Forming Units (SFU) become no longer precisely countable: “too numerous to count is shown by a star; ( C ) While at high numbers spot counts tend to deviate from linearity due to crowding, in lower numbers they followed a liner relationship between cell numbers plated and SFU counted, in this case with an R 2 value of 0.9939. Based on the regression line shown in red, the pan-IgG SFU frequency has been calculated to be, in the example shown here, 13,800 pan-IgG SFU per one million PBMC.
Figure Legend Snippet: Calculating frequencies for pan IgG-SFU/million PBMC illustrated. Polyclonally stimulated PBMC were plated in the specified cell numbers in quadruplicate wells, and a Three Color ImmunoSpot ® assay was performed for Ig classes pan-IgG, M and A. ( A ) Raw images for the IgG color plane are shown with the cell numbers plated per well specified; ( B ) the mean spot count for each cell concentration, with the SD for the quadruplicate wells tested is plotted vs. the cell numbers plated. As the spots at high ASC densities start crowding (plus when the number of ASC is high, there is an “ELISA” effect with analyte captured from the supernatant causing a high background carpet staining), for high cell numbers Spot Forming Units (SFU) become no longer precisely countable: “too numerous to count is shown by a star; ( C ) While at high numbers spot counts tend to deviate from linearity due to crowding, in lower numbers they followed a liner relationship between cell numbers plated and SFU counted, in this case with an R 2 value of 0.9939. Based on the regression line shown in red, the pan-IgG SFU frequency has been calculated to be, in the example shown here, 13,800 pan-IgG SFU per one million PBMC.

Techniques Used: Concentration Assay, Staining

Effect of cryopreservation on IgG subclass production by B cells. The legend to Figure 5 applies except that a four color B cell ImmunoSpot ® assay was performed measuring IgG1 ( A ), IgG2 ( B ), IgG3 ( C ), and IgG4 ( D ) on the serially diluted PBMC. Results obtained for 15 PBMC donors tested are shown.
Figure Legend Snippet: Effect of cryopreservation on IgG subclass production by B cells. The legend to Figure 5 applies except that a four color B cell ImmunoSpot ® assay was performed measuring IgG1 ( A ), IgG2 ( B ), IgG3 ( C ), and IgG4 ( D ) on the serially diluted PBMC. Results obtained for 15 PBMC donors tested are shown.

Techniques Used:

Effect of cryopreservation on Ig class production by B cells. Freshly isolated PBMC were polyclonally stimulated for four days and then tested in a three color B cell ImmunoSpot ® assay, with serial dilution of the cells in four replicate wells, (“Fresh”), as illustrated in Figure 4 . The same PBMC were cryopreserved, thawed, and then polyclonally stimulated, seeded and tested as above (“Frozen”). The latter cells were cryopreserved at the end of the four day polyclonal stimulation culture, when B cell blasts have been engaged, then thawed, and seeded and tested as above (“Blasts”). The relationship of Fresh/Frozen and Blast cells is graphically illustrated in Figure S1 . The results obtained testing 15 PBMC donors are shown here for pan-IgG ( A ), IgM ( B ), and IgA ( C ). The SFU counts per million PBMC have been established as specified in Figure 4 . For each donor, as defined by color in the insert, the mean spot counts are connected by the corresponding color coded lines.
Figure Legend Snippet: Effect of cryopreservation on Ig class production by B cells. Freshly isolated PBMC were polyclonally stimulated for four days and then tested in a three color B cell ImmunoSpot ® assay, with serial dilution of the cells in four replicate wells, (“Fresh”), as illustrated in Figure 4 . The same PBMC were cryopreserved, thawed, and then polyclonally stimulated, seeded and tested as above (“Frozen”). The latter cells were cryopreserved at the end of the four day polyclonal stimulation culture, when B cell blasts have been engaged, then thawed, and seeded and tested as above (“Blasts”). The relationship of Fresh/Frozen and Blast cells is graphically illustrated in Figure S1 . The results obtained testing 15 PBMC donors are shown here for pan-IgG ( A ), IgM ( B ), and IgA ( C ). The SFU counts per million PBMC have been established as specified in Figure 4 . For each donor, as defined by color in the insert, the mean spot counts are connected by the corresponding color coded lines.

Techniques Used: Isolation, Serial Dilution

Related Articles

Enzyme-linked Immunospot:

Article Title: An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi
Article Snippet: .. ELISPOT Assays with PBMC All PBMC samples were assayed using the human IFN-γ ImmunoSpot kit by Cellular Technology Limited (OH, USA) per the manufacturer’s instruction. ..

Article Title: ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques
Article Snippet: .. NHPs – IFN-γ ELISpot assay of PBMCs was performed using the ImmunoSpot® Human IFN- γ Single-Color Enzymatic ELISpot Assay Kit according to the manufacturer’s protocol (Cellular Technology Limited). .. PBMCs were plated at a concentration of 100,000 cells per well and were stimulated with four contiguous peptide pools spanning the length of the SARS-CoV-2 spike protein sequence at a concentration of 2 μg/mL per peptide (Mimotopes).

Article Title: Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4
Article Snippet: .. Co-culture of MDSC or DRhi cells with PBMCs For Enzyme-Linked ImmunoSpot Assay (ELISPOT), 1 × 105 sorted MDSC or HLD DRhi cells were co-cultured with autologous 2 × 105 PBMCs in the presence of 1 μg/ml peptide pool of CMVpp65 (CMVpp65) (NIH AIDS Reagent Program) for 18–20 hrs. .. For ELISA and intracellular staining, 2 × 105 sorted MDSC or HLD DRhi cells were co-cultured with autologous 4 × 105 PBMCs in the presence of CMVpp65 for 48 hrs, supernatants were frozen and cells stained for intracellular IFNγ.

Cell Culture:

Article Title: Severe H7N9 Infection Is Associated with Decreased Antigen-Presenting Capacity of CD14+ Cells
Article Snippet: .. PBMCs were cultured with polyI:C (20 ng/ml) for 24 h, and the positive cells enumerated by an ImmunoSpot S5 Macro Analyzer (C.T.L., Shaker Heights, OH, USA) were expressed as numbers of IFN-γ or IL-17 spot-forming units per well. ..

Co-Culture Assay:

Article Title: Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4
Article Snippet: .. Co-culture of MDSC or DRhi cells with PBMCs For Enzyme-Linked ImmunoSpot Assay (ELISPOT), 1 × 105 sorted MDSC or HLD DRhi cells were co-cultured with autologous 2 × 105 PBMCs in the presence of 1 μg/ml peptide pool of CMVpp65 (CMVpp65) (NIH AIDS Reagent Program) for 18–20 hrs. .. For ELISA and intracellular staining, 2 × 105 sorted MDSC or HLD DRhi cells were co-cultured with autologous 4 × 105 PBMCs in the presence of CMVpp65 for 48 hrs, supernatants were frozen and cells stained for intracellular IFNγ.

CTL Assay:

Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays
Article Snippet: .. Thawing and Resting of Cryopreserved PBMC Cryopreserved human PBMC of 25 donors were acquired from a library of PBMC (ePBMC, CTL‑CP1) offered by Cellular Technologies Ltd. (CTL, Shaker Heights, OH, USA). ..

Article Title: Nuclease-resistant immunostimulatory phosphodiester CpG oligodeoxynucleotides as human Toll-like receptor 9 agonists
Article Snippet: .. Measurement of IL-6 release from PBMC and CAL-1 After thawing, PBMCs were seeded in serum free medium, CTL-test medium (Cellular Technology Limited), at a density of 1.0 × 106 cells/mL. ..

Article Title: Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify
Article Snippet: .. For polyclonal stimulation, freshly thawed PBMC were resuspended in CTL-Test B™-media (CTLTB-010, Cellular Technology Ltd., Cleveland, OH, USA) supplemented with polyclonal B-cell stimulator (CTL-BPOLY200) that contains R848 and human IL-2 and is part of the kit. ..

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    Cellular Technology Ltd pbmcs
    HIV <t>MDSC</t> control CMV specific IFNγ production from CD4 + CX3CR1 + T cells. HIV MDSC were expanded, sorted and cultured with autologous freshly isolated <t>PBMCs</t> as in Fig. 2 . Cells were sorted and cultured for 48 hrs, Brefeldin A was added for the last 5 hrs of culture. Cells were surface stained with anti-CD14, -CD3, -CD4 and –CX3CR1, fixed, permeabilized and stained using anti-IFNγ for intracellular IFNγ. (a) Representative flow cytometry histograms are shown. (b) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. (c) Supernatants were collected and the quantity of IFNγ determined by ELISA. (d) Blood obtained from HIV(−) (n = 7) and HIV(+) individuals on ART (HIV+) (n = 12) was stained with anti-CD3,-CD19,-CD11b,-CD33,-CD14, -HLA DR Abs, cells were analyzed as CD3 − CD19 − CD11b + CD33 + CD14 + HLA DR −/lo by flow cytometry. Percentages of MDSCs are shown. (e) Representative dot plot with HLA DR −/lo region is shown. For ( d ) Each dot in the plots depict data of each individual donor, the plots include observations from 25 th to 75 th percentile. The horizontal line represents the median value. For ( b and c ), histograms are presented as mean+/−SD; n = 3 donors; *p
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    Cellular Technology Ltd interferon γ enzyme linked immunospot peripheral blood mononuclear cells
    Cellular immunity to pandemic influenza virus (pH1N1) antigens in donor baseline peripheral blood mononuclear cells. Responses were measured by interferon (IFN)-γ enzyme-linked <t>immunospot</t> (ELISPOT). Stimulation used 25 pH1N1 2009 peptide pools (see Methods and Supplementary Table 1), at 2 μg/mL final concentration of each peptide in the culture. A standard CEF peptide pool and phytohemagglutinin were used as positive controls, and media alone was used as negative control. Spots were counted 24 hours later. Bars show responses for individual donors as IFN-γ spot-forming cells (SFC)/10 6 cells above background. Colored segments of bars indicate antigen recognized. (A) Responses at the baseline time point for 153 donors (not all of whom were evaluable for infection because some lacked follow-up serum samples). (B and C) Responses at the baseline time point in those donors later infected with pH1N1, grouped for symptoms of differing severity. Due to limited availability of cells, certain infected donors were not tested with the 25 peptide pools and thus are not shown. (B) Infected donors with influenza-like illness (ILI) symptoms. (C) Donors with milder or no symptoms. Total IFN-γ ELISPOT response did not differ significantly between the ILI and mild/asymptomatic groups, P = .127 by 2-tailed Student t test.
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    Cellular Technology Ltd regulatory t cell treg assessment frozen pbmcs
    <t>Regulatory</t> T cells <t>(Treg)</t> in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. a Gating strategy used to identify blood Treg cells within the CD4 + and CD8 + T-cell compartment. b Percentage of CD4 + Treg cells in different patient groups (TKIs group, n = 33; TKIs plus IFN-α group, n = 8) and in healthy controls (n = 20). c Frequency of CD4 + Treg cells in patients with CML receiving imatinib (n = 26) or 2nd generation TKIs (n = 7). d Frequency of CD8 + Treg cells in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. The p values in the figure reflect statistically significant differences among study groups
    Regulatory T Cell Treg Assessment Frozen Pbmcs, supplied by Cellular Technology Ltd, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/regulatory t cell treg assessment frozen pbmcs/product/Cellular Technology Ltd
    Average 94 stars, based on 1 article reviews
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    85
    Cellular Technology Ltd mumps antigen cryopreserved pbmc
    CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The <t>PBMC</t> and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, <t>non-cryopreserved</t> PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.
    Mumps Antigen Cryopreserved Pbmc, supplied by Cellular Technology Ltd, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HIV MDSC control CMV specific IFNγ production from CD4 + CX3CR1 + T cells. HIV MDSC were expanded, sorted and cultured with autologous freshly isolated PBMCs as in Fig. 2 . Cells were sorted and cultured for 48 hrs, Brefeldin A was added for the last 5 hrs of culture. Cells were surface stained with anti-CD14, -CD3, -CD4 and –CX3CR1, fixed, permeabilized and stained using anti-IFNγ for intracellular IFNγ. (a) Representative flow cytometry histograms are shown. (b) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. (c) Supernatants were collected and the quantity of IFNγ determined by ELISA. (d) Blood obtained from HIV(−) (n = 7) and HIV(+) individuals on ART (HIV+) (n = 12) was stained with anti-CD3,-CD19,-CD11b,-CD33,-CD14, -HLA DR Abs, cells were analyzed as CD3 − CD19 − CD11b + CD33 + CD14 + HLA DR −/lo by flow cytometry. Percentages of MDSCs are shown. (e) Representative dot plot with HLA DR −/lo region is shown. For ( d ) Each dot in the plots depict data of each individual donor, the plots include observations from 25 th to 75 th percentile. The horizontal line represents the median value. For ( b and c ), histograms are presented as mean+/−SD; n = 3 donors; *p

    Journal: Scientific Reports

    Article Title: Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4

    doi: 10.1038/srep44485

    Figure Lengend Snippet: HIV MDSC control CMV specific IFNγ production from CD4 + CX3CR1 + T cells. HIV MDSC were expanded, sorted and cultured with autologous freshly isolated PBMCs as in Fig. 2 . Cells were sorted and cultured for 48 hrs, Brefeldin A was added for the last 5 hrs of culture. Cells were surface stained with anti-CD14, -CD3, -CD4 and –CX3CR1, fixed, permeabilized and stained using anti-IFNγ for intracellular IFNγ. (a) Representative flow cytometry histograms are shown. (b) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. (c) Supernatants were collected and the quantity of IFNγ determined by ELISA. (d) Blood obtained from HIV(−) (n = 7) and HIV(+) individuals on ART (HIV+) (n = 12) was stained with anti-CD3,-CD19,-CD11b,-CD33,-CD14, -HLA DR Abs, cells were analyzed as CD3 − CD19 − CD11b + CD33 + CD14 + HLA DR −/lo by flow cytometry. Percentages of MDSCs are shown. (e) Representative dot plot with HLA DR −/lo region is shown. For ( d ) Each dot in the plots depict data of each individual donor, the plots include observations from 25 th to 75 th percentile. The horizontal line represents the median value. For ( b and c ), histograms are presented as mean+/−SD; n = 3 donors; *p

    Article Snippet: Co-culture of MDSC or DRhi cells with PBMCs For Enzyme-Linked ImmunoSpot Assay (ELISPOT), 1 × 105 sorted MDSC or HLD DRhi cells were co-cultured with autologous 2 × 105 PBMCs in the presence of 1 μg/ml peptide pool of CMVpp65 (CMVpp65) (NIH AIDS Reagent Program) for 18–20 hrs.

    Techniques: Cell Culture, Isolation, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    IL-27 regulates B7-H4 expression on HIV expanded MDSC. (a) To determine if IL-27 regulates B7-H4 expression, PBMCs from HIV(−) donors were cultured with or without rIL-27 (5 ng/ml) for 72 hrs. Cells cultured in the presence of non-infectious HIV BaL (p24, 12 ng/10 7 cells) or rIL-10 (5 ng/ml) served as additional controls. Whole cell lysates of PBMCs were prepared and immunoblotted with Abs to GAPDH as loading control and B7-H4. (ai) Immunoblot shown is representative of 4 donors; cells cultured with rIL10 and rIL-27 were run in duplicates. (aii) The histogram bar graph shows mean+/−SD of the densitometric analysis for B7-H4 (n = 4). (b–d) Effect of IL-6, IL-10 and IL-27 on MDSC and B7-H4 + HIV MDSC was determined. PBMCs of healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells) in the presence or absence of neutralizing anti-IL6, -IL-10 or –IL-27 or respective isotype antibodies for 5 days. Cells were stained with anti-CD11b, -CD33, -CD14, -HLA DR and –B7-H4 Abs and analyzed using flow cytometry. (b) %B7-H4 + MDSC (bi) and MFI of B7-H4 on MDSC (bii) is shown in presence of anti-IL-6 or-IL-10 antibodies. (c) %B7-H4 + MDSC (ci) and MFI of B7-H4 on MDSC (cii) is shown in presence of anti-IL-27 antibody. (d) %MDSC is shown in presence of anti-IL-6 and –IL-10 (di) and anti-IL-27 (dii) antibodies. Histograms are presented as mean+/−SD; n = 4 donors. (e) Regulation of immunity in HIV/CMV co-infection: HIV/CMV co-infected individuals on ART with suppressed HIV replication and recovered CD4 + T cell numbers have increased IL-27 and reduced MDSC, IL-27 induces (1) B7-H4 on MDSC which binds to CMV-CD4 + T cells and (2) mediates CD4 + IL-10 + T cells, these collectively regulates T cell activation such that IFNγ is produced that is sufficient to control CMV replication without inducing inflammation (right side of straight line) . HIV/CMV co-infected ART naïve individuals with replicating virus have increased IL-6 that induces MDSC expansion and MDSC contributes to immune suppression (left side of straight line) observed during chronic disease stage. *p

    Journal: Scientific Reports

    Article Title: Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4

    doi: 10.1038/srep44485

    Figure Lengend Snippet: IL-27 regulates B7-H4 expression on HIV expanded MDSC. (a) To determine if IL-27 regulates B7-H4 expression, PBMCs from HIV(−) donors were cultured with or without rIL-27 (5 ng/ml) for 72 hrs. Cells cultured in the presence of non-infectious HIV BaL (p24, 12 ng/10 7 cells) or rIL-10 (5 ng/ml) served as additional controls. Whole cell lysates of PBMCs were prepared and immunoblotted with Abs to GAPDH as loading control and B7-H4. (ai) Immunoblot shown is representative of 4 donors; cells cultured with rIL10 and rIL-27 were run in duplicates. (aii) The histogram bar graph shows mean+/−SD of the densitometric analysis for B7-H4 (n = 4). (b–d) Effect of IL-6, IL-10 and IL-27 on MDSC and B7-H4 + HIV MDSC was determined. PBMCs of healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells) in the presence or absence of neutralizing anti-IL6, -IL-10 or –IL-27 or respective isotype antibodies for 5 days. Cells were stained with anti-CD11b, -CD33, -CD14, -HLA DR and –B7-H4 Abs and analyzed using flow cytometry. (b) %B7-H4 + MDSC (bi) and MFI of B7-H4 on MDSC (bii) is shown in presence of anti-IL-6 or-IL-10 antibodies. (c) %B7-H4 + MDSC (ci) and MFI of B7-H4 on MDSC (cii) is shown in presence of anti-IL-27 antibody. (d) %MDSC is shown in presence of anti-IL-6 and –IL-10 (di) and anti-IL-27 (dii) antibodies. Histograms are presented as mean+/−SD; n = 4 donors. (e) Regulation of immunity in HIV/CMV co-infection: HIV/CMV co-infected individuals on ART with suppressed HIV replication and recovered CD4 + T cell numbers have increased IL-27 and reduced MDSC, IL-27 induces (1) B7-H4 on MDSC which binds to CMV-CD4 + T cells and (2) mediates CD4 + IL-10 + T cells, these collectively regulates T cell activation such that IFNγ is produced that is sufficient to control CMV replication without inducing inflammation (right side of straight line) . HIV/CMV co-infected ART naïve individuals with replicating virus have increased IL-6 that induces MDSC expansion and MDSC contributes to immune suppression (left side of straight line) observed during chronic disease stage. *p

    Article Snippet: Co-culture of MDSC or DRhi cells with PBMCs For Enzyme-Linked ImmunoSpot Assay (ELISPOT), 1 × 105 sorted MDSC or HLD DRhi cells were co-cultured with autologous 2 × 105 PBMCs in the presence of 1 μg/ml peptide pool of CMVpp65 (CMVpp65) (NIH AIDS Reagent Program) for 18–20 hrs.

    Techniques: Expressing, Cell Culture, Staining, Flow Cytometry, Cytometry, Infection, Activation Assay, Produced

    In vitro expanded HIV MDSC are functionally similar to MDSC in HIV-infected individuals and modulate CMV specific T cell response. PBMCs of CMV(+) HIV(−) donors were cultured without (PBMCs) or with inactivated HIV BaL (HIV PBMCs) (p24 Ag, 12 ng/10 7 cells). After 5 days, cells were stained with anti-CD11b, -CD33, CD14, HLA DR. ( a and b ) CD14 + HLA DR −/lo MDSC were depleted from HIV PBMCs. HIV PBMCs, HIV PBMCs and MDSC depleted HIV PBMCs (HIV MDSC dep) were cultured with or without CMVpp65 for 24 and 48 hrs. The amounts of IL-2 ( a ) and IFNγ ( b ) were determined in culture supernatants at 24 and 48 hrs, respectively. Net IL-2 or IFNγ was calculated as CMVpp65 – Control. ( c and d ) PBMCs were cultured and stained as above, DR hi and MDSC were sorted. (ci and cii) Representative dot plots show HLA DR vs CD14 in control cells before sort and after sort (Left panel) and in HIV BaL treated cells before sort and after sort (Right panel) . Cells isolated are > 98% pure. ( d and e ) Isolated HIV DR hi and MDSC (5 × 10 4 ) were cultured overnight with autologous freshly isolated PBMCs (1 × 10 5 ) on ELISPOT plates with or without CMVpp65 to determine frequency of ( d ) IFNγ producing cells and ( e ) IL-10 producing cells. Histograms are presented as mean+/−SD; n = 3 donors; *p

    Journal: Scientific Reports

    Article Title: Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4

    doi: 10.1038/srep44485

    Figure Lengend Snippet: In vitro expanded HIV MDSC are functionally similar to MDSC in HIV-infected individuals and modulate CMV specific T cell response. PBMCs of CMV(+) HIV(−) donors were cultured without (PBMCs) or with inactivated HIV BaL (HIV PBMCs) (p24 Ag, 12 ng/10 7 cells). After 5 days, cells were stained with anti-CD11b, -CD33, CD14, HLA DR. ( a and b ) CD14 + HLA DR −/lo MDSC were depleted from HIV PBMCs. HIV PBMCs, HIV PBMCs and MDSC depleted HIV PBMCs (HIV MDSC dep) were cultured with or without CMVpp65 for 24 and 48 hrs. The amounts of IL-2 ( a ) and IFNγ ( b ) were determined in culture supernatants at 24 and 48 hrs, respectively. Net IL-2 or IFNγ was calculated as CMVpp65 – Control. ( c and d ) PBMCs were cultured and stained as above, DR hi and MDSC were sorted. (ci and cii) Representative dot plots show HLA DR vs CD14 in control cells before sort and after sort (Left panel) and in HIV BaL treated cells before sort and after sort (Right panel) . Cells isolated are > 98% pure. ( d and e ) Isolated HIV DR hi and MDSC (5 × 10 4 ) were cultured overnight with autologous freshly isolated PBMCs (1 × 10 5 ) on ELISPOT plates with or without CMVpp65 to determine frequency of ( d ) IFNγ producing cells and ( e ) IL-10 producing cells. Histograms are presented as mean+/−SD; n = 3 donors; *p

    Article Snippet: Co-culture of MDSC or DRhi cells with PBMCs For Enzyme-Linked ImmunoSpot Assay (ELISPOT), 1 × 105 sorted MDSC or HLD DRhi cells were co-cultured with autologous 2 × 105 PBMCs in the presence of 1 μg/ml peptide pool of CMVpp65 (CMVpp65) (NIH AIDS Reagent Program) for 18–20 hrs.

    Techniques: In Vitro, Infection, Cell Culture, Staining, Isolation, Enzyme-linked Immunospot

    B7-H4 down regulates pAkt to regulate CMV induced T cell activation. To determine the effect of HIV MDSC on CMVpp65 induced pZap70 or pAkt, PBMCs of CMV(+) HIV(−) donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells). After 5 days, cells were stained with anti -CD11b, -CD33, CD14, HLA DR; DR hi and MDSC were sorted. (a–c) Sorted cells and freshly isolated autologous PBMCs were cultured in a ratio of 1:2 (1 sorted cell: 2 PBMCs) with or without CMVpp65 for 15 min. pZap70 was determined as detailed in Methods section. (a) Representative flow cytometry histogram plot is shown, (b) pZap70 expressed as %Net CD3 + CD4 + pZap70 + cells and (c) MFI of pZap70 in CD3 + CD4 + cells was determined. (d–f) Sorted MDSC were transfected with control or B7-H4 siRNA (75 nM) using Lipofectamine RNAiMAX reagent (Invitrogen); HIV DR hi cells were cultured in presence of Lipofectamine RNAiMAX reagent used for transfecting MDSC. After 48–72 hrs, transfected cells and freshly isolated autologous PBMCs were cultured in a ratio of 1:2 (1 sorted cell: 2 PBMCs) with or without CMVpp65 peptide pool for 15–30 min pAkt was determined as above. (d) Representative flow cytometry histogram plot is shown, (e) pAkt expressed as %Net CD3 + CD4 + pAkt + cells and (f) MFI of pAkt in CD3 + CD4 + cells was determined. Histograms are presented as mean+/−SD; n = 4 donors. *p

    Journal: Scientific Reports

    Article Title: Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4

    doi: 10.1038/srep44485

    Figure Lengend Snippet: B7-H4 down regulates pAkt to regulate CMV induced T cell activation. To determine the effect of HIV MDSC on CMVpp65 induced pZap70 or pAkt, PBMCs of CMV(+) HIV(−) donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells). After 5 days, cells were stained with anti -CD11b, -CD33, CD14, HLA DR; DR hi and MDSC were sorted. (a–c) Sorted cells and freshly isolated autologous PBMCs were cultured in a ratio of 1:2 (1 sorted cell: 2 PBMCs) with or without CMVpp65 for 15 min. pZap70 was determined as detailed in Methods section. (a) Representative flow cytometry histogram plot is shown, (b) pZap70 expressed as %Net CD3 + CD4 + pZap70 + cells and (c) MFI of pZap70 in CD3 + CD4 + cells was determined. (d–f) Sorted MDSC were transfected with control or B7-H4 siRNA (75 nM) using Lipofectamine RNAiMAX reagent (Invitrogen); HIV DR hi cells were cultured in presence of Lipofectamine RNAiMAX reagent used for transfecting MDSC. After 48–72 hrs, transfected cells and freshly isolated autologous PBMCs were cultured in a ratio of 1:2 (1 sorted cell: 2 PBMCs) with or without CMVpp65 peptide pool for 15–30 min pAkt was determined as above. (d) Representative flow cytometry histogram plot is shown, (e) pAkt expressed as %Net CD3 + CD4 + pAkt + cells and (f) MFI of pAkt in CD3 + CD4 + cells was determined. Histograms are presented as mean+/−SD; n = 4 donors. *p

    Article Snippet: Co-culture of MDSC or DRhi cells with PBMCs For Enzyme-Linked ImmunoSpot Assay (ELISPOT), 1 × 105 sorted MDSC or HLD DRhi cells were co-cultured with autologous 2 × 105 PBMCs in the presence of 1 μg/ml peptide pool of CMVpp65 (CMVpp65) (NIH AIDS Reagent Program) for 18–20 hrs.

    Techniques: Activation Assay, Cell Culture, Staining, Isolation, Flow Cytometry, Cytometry, Transfection

    HIV MDSC overexpress the inhibitory ligand B7-H4 and regulate CMV T cell activation. PBMCs from healthy donors were cultured in the presence or absence of HIV BaL . After 5 days, cells were stained using anti-CD11b, -CD33, -CD14, -HLA DR and -B7-H1/-B7-H3/-PD-L2/-ICOS-L or –B7-H4. (a and b ) Expression of B7-H1, B7-H3, PD-L2, ICOS-L or B7-H4 was determined by flow cytometry and fold expression of each ligand calculated: MDSC (Control cells - HIV BaL stimulated cells)/DR hi (Control cell - HIV BaL stimulated cells). (a) Mean values ± SD from 5 donors are shown (b) Representative flow cytometry histogram plot is shown. (c) Net Mean Fluorescence Intensity (MFI) of respective inhibitory ligands in DR hi and MDSC was determined: MFI in Control cells – MFI in HIV BaL stimulated. Mean ± SD from 5 donors are shown. ( d and e ) PBMCs of CMV(+) HIV(−) healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells). After 5 days, cells were stained with anti- CD11b, -CD33, CD14, HLA DR; DR hi and MDSC were sorted. MDSC were transfected with control or B7-H4 siRNA (75 nM) using Lipofectamine RNAiMAX reagent (Invitrogen). HIV DR hi cells were cultured in the presence of Lipofectamine RNAiMAX reagent used for transfecting MDSC. After 48–72 hrs, cell lysates of control and B7-H4 siRNA transfected cells were prepared and immunoblotted using anti-GAPDH and –B7-H4 Ab to determine expression of B7-H4. 2.5 × 10 4 transfected cells were cultured with 5 × 10 4 freshly isolated autologous PBMCs in the presence or absence of CMVpp65. (d) After 18 hrs, IL-2 and (e) after 48 hrs IFNγ was determined in culture supernatants by ELISA; partial knockdown of B7-H4 is shown in d. (f and g) PBMCs of CMV(+) HIV(−) healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells) and MDSC isolated as above. 0.05 × 10 6 MDSC were cultured with 0.1 × 10 6 autologous PBMCs in presence or absence of CMVpp65 with or without rB7-H4 (10 ng/ml). (f) After 18 hrs, IL-2 and (g) after 48 hrs IFNγ was determined in culture supernatants by ELISA. Mean+/−SD are shown for 3 healthy donors. *p

    Journal: Scientific Reports

    Article Title: Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4

    doi: 10.1038/srep44485

    Figure Lengend Snippet: HIV MDSC overexpress the inhibitory ligand B7-H4 and regulate CMV T cell activation. PBMCs from healthy donors were cultured in the presence or absence of HIV BaL . After 5 days, cells were stained using anti-CD11b, -CD33, -CD14, -HLA DR and -B7-H1/-B7-H3/-PD-L2/-ICOS-L or –B7-H4. (a and b ) Expression of B7-H1, B7-H3, PD-L2, ICOS-L or B7-H4 was determined by flow cytometry and fold expression of each ligand calculated: MDSC (Control cells - HIV BaL stimulated cells)/DR hi (Control cell - HIV BaL stimulated cells). (a) Mean values ± SD from 5 donors are shown (b) Representative flow cytometry histogram plot is shown. (c) Net Mean Fluorescence Intensity (MFI) of respective inhibitory ligands in DR hi and MDSC was determined: MFI in Control cells – MFI in HIV BaL stimulated. Mean ± SD from 5 donors are shown. ( d and e ) PBMCs of CMV(+) HIV(−) healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells). After 5 days, cells were stained with anti- CD11b, -CD33, CD14, HLA DR; DR hi and MDSC were sorted. MDSC were transfected with control or B7-H4 siRNA (75 nM) using Lipofectamine RNAiMAX reagent (Invitrogen). HIV DR hi cells were cultured in the presence of Lipofectamine RNAiMAX reagent used for transfecting MDSC. After 48–72 hrs, cell lysates of control and B7-H4 siRNA transfected cells were prepared and immunoblotted using anti-GAPDH and –B7-H4 Ab to determine expression of B7-H4. 2.5 × 10 4 transfected cells were cultured with 5 × 10 4 freshly isolated autologous PBMCs in the presence or absence of CMVpp65. (d) After 18 hrs, IL-2 and (e) after 48 hrs IFNγ was determined in culture supernatants by ELISA; partial knockdown of B7-H4 is shown in d. (f and g) PBMCs of CMV(+) HIV(−) healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells) and MDSC isolated as above. 0.05 × 10 6 MDSC were cultured with 0.1 × 10 6 autologous PBMCs in presence or absence of CMVpp65 with or without rB7-H4 (10 ng/ml). (f) After 18 hrs, IL-2 and (g) after 48 hrs IFNγ was determined in culture supernatants by ELISA. Mean+/−SD are shown for 3 healthy donors. *p

    Article Snippet: Co-culture of MDSC or DRhi cells with PBMCs For Enzyme-Linked ImmunoSpot Assay (ELISPOT), 1 × 105 sorted MDSC or HLD DRhi cells were co-cultured with autologous 2 × 105 PBMCs in the presence of 1 μg/ml peptide pool of CMVpp65 (CMVpp65) (NIH AIDS Reagent Program) for 18–20 hrs.

    Techniques: Activation Assay, Cell Culture, Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence, Transfection, Isolation, Enzyme-linked Immunosorbent Assay

    MDSC regulate CD4 + CX3CR1 + IFNγ + cells in HIV/CMV co-infected individuals. (a–c) All donors were CMV(+) and either HIV-infected (HIV+) or HIV–uninfected (HIV−). PBMCs of HIV(−) and HIV(+) donors were cultured with or without CMVpp65 for 72 hrs. Brefeldin A was added for the last 5 hrs of culture. Cells were surface stained with anti-CD14, -CD3, -CD4 and –CX3CR1, fixed, permeabilized and stained using anti-IFNγ for intracellular IFNγ. ( a ) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. The plots include observations from 25th to 75th percentile; the horizontal line represents the median value. ( b ) Percentage of IFNγ + cells were determined in CD14 − CD3 + CD4 + CX3CR1 + (open histograms) and CD14 − CD3 + CD4 + CX3CR1 − (shaded histograms) subsets. ( c ) Representative dot plots showing CD4 + IFNγ + cells gated on CD14 − CD3 + CD4 + CX3CR1 + cells are shown from HIV-uninfected (ci HIV-ve) and HIV-infected (cii HIV+ve) individual. (d and e) Freshly isolated PBMCs from CMV(+) HIV-infected individuals on ART and with suppressed viral replication were stained with anti-CD14 and –HLA DR antibodies; CD14 + HLA DR −/lo MDSC were depleted from PBMCs by flow cytometry. Whole PBMC (PBMC) and MDSC depleted PBMC (PBMC-MDSC) were cultured with or without CMVpp65 for 72 hrs as above. Culture supernatant was stored and cells were stained as detailed above. ( d ) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. ( e ) Amount of IFNγ in the culture supernatants was determined by ELISA. The histograms in b show mean values+/−SD; n = 8 donors. For all other graphs each dot represents an individual donor; *p

    Journal: Scientific Reports

    Article Title: Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4

    doi: 10.1038/srep44485

    Figure Lengend Snippet: MDSC regulate CD4 + CX3CR1 + IFNγ + cells in HIV/CMV co-infected individuals. (a–c) All donors were CMV(+) and either HIV-infected (HIV+) or HIV–uninfected (HIV−). PBMCs of HIV(−) and HIV(+) donors were cultured with or without CMVpp65 for 72 hrs. Brefeldin A was added for the last 5 hrs of culture. Cells were surface stained with anti-CD14, -CD3, -CD4 and –CX3CR1, fixed, permeabilized and stained using anti-IFNγ for intracellular IFNγ. ( a ) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. The plots include observations from 25th to 75th percentile; the horizontal line represents the median value. ( b ) Percentage of IFNγ + cells were determined in CD14 − CD3 + CD4 + CX3CR1 + (open histograms) and CD14 − CD3 + CD4 + CX3CR1 − (shaded histograms) subsets. ( c ) Representative dot plots showing CD4 + IFNγ + cells gated on CD14 − CD3 + CD4 + CX3CR1 + cells are shown from HIV-uninfected (ci HIV-ve) and HIV-infected (cii HIV+ve) individual. (d and e) Freshly isolated PBMCs from CMV(+) HIV-infected individuals on ART and with suppressed viral replication were stained with anti-CD14 and –HLA DR antibodies; CD14 + HLA DR −/lo MDSC were depleted from PBMCs by flow cytometry. Whole PBMC (PBMC) and MDSC depleted PBMC (PBMC-MDSC) were cultured with or without CMVpp65 for 72 hrs as above. Culture supernatant was stored and cells were stained as detailed above. ( d ) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. ( e ) Amount of IFNγ in the culture supernatants was determined by ELISA. The histograms in b show mean values+/−SD; n = 8 donors. For all other graphs each dot represents an individual donor; *p

    Article Snippet: Co-culture of MDSC or DRhi cells with PBMCs For Enzyme-Linked ImmunoSpot Assay (ELISPOT), 1 × 105 sorted MDSC or HLD DRhi cells were co-cultured with autologous 2 × 105 PBMCs in the presence of 1 μg/ml peptide pool of CMVpp65 (CMVpp65) (NIH AIDS Reagent Program) for 18–20 hrs.

    Techniques: Infection, Cell Culture, Staining, Flow Cytometry, Cytometry, Isolation, Enzyme-linked Immunosorbent Assay

    Cellular immunity to pandemic influenza virus (pH1N1) antigens in donor baseline peripheral blood mononuclear cells. Responses were measured by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT). Stimulation used 25 pH1N1 2009 peptide pools (see Methods and Supplementary Table 1), at 2 μg/mL final concentration of each peptide in the culture. A standard CEF peptide pool and phytohemagglutinin were used as positive controls, and media alone was used as negative control. Spots were counted 24 hours later. Bars show responses for individual donors as IFN-γ spot-forming cells (SFC)/10 6 cells above background. Colored segments of bars indicate antigen recognized. (A) Responses at the baseline time point for 153 donors (not all of whom were evaluable for infection because some lacked follow-up serum samples). (B and C) Responses at the baseline time point in those donors later infected with pH1N1, grouped for symptoms of differing severity. Due to limited availability of cells, certain infected donors were not tested with the 25 peptide pools and thus are not shown. (B) Infected donors with influenza-like illness (ILI) symptoms. (C) Donors with milder or no symptoms. Total IFN-γ ELISPOT response did not differ significantly between the ILI and mild/asymptomatic groups, P = .127 by 2-tailed Student t test.

    Journal: Open Forum Infectious Diseases

    Article Title: Surveillance Study of Influenza Occurrence and Immunity in a Wisconsin Cohort During the 2009 Pandemic

    doi: 10.1093/ofid/ofx023

    Figure Lengend Snippet: Cellular immunity to pandemic influenza virus (pH1N1) antigens in donor baseline peripheral blood mononuclear cells. Responses were measured by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT). Stimulation used 25 pH1N1 2009 peptide pools (see Methods and Supplementary Table 1), at 2 μg/mL final concentration of each peptide in the culture. A standard CEF peptide pool and phytohemagglutinin were used as positive controls, and media alone was used as negative control. Spots were counted 24 hours later. Bars show responses for individual donors as IFN-γ spot-forming cells (SFC)/10 6 cells above background. Colored segments of bars indicate antigen recognized. (A) Responses at the baseline time point for 153 donors (not all of whom were evaluable for infection because some lacked follow-up serum samples). (B and C) Responses at the baseline time point in those donors later infected with pH1N1, grouped for symptoms of differing severity. Due to limited availability of cells, certain infected donors were not tested with the 25 peptide pools and thus are not shown. (B) Infected donors with influenza-like illness (ILI) symptoms. (C) Donors with milder or no symptoms. Total IFN-γ ELISPOT response did not differ significantly between the ILI and mild/asymptomatic groups, P = .127 by 2-tailed Student t test.

    Article Snippet: Interferon-γ Enzyme-Linked Immunospot Peripheral blood mononuclear cells were cultured in triplicate for 24 hours with stimulating peptide pools containing 2 µg/mL of each peptide, in anti-interferon (IFN)-γ-coated plates.

    Techniques: Enzyme-linked Immunospot, Concentration Assay, Negative Control, Infection

    Regulatory T cells (Treg) in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. a Gating strategy used to identify blood Treg cells within the CD4 + and CD8 + T-cell compartment. b Percentage of CD4 + Treg cells in different patient groups (TKIs group, n = 33; TKIs plus IFN-α group, n = 8) and in healthy controls (n = 20). c Frequency of CD4 + Treg cells in patients with CML receiving imatinib (n = 26) or 2nd generation TKIs (n = 7). d Frequency of CD8 + Treg cells in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. The p values in the figure reflect statistically significant differences among study groups

    Journal: Journal of Translational Medicine

    Article Title: Flow cytometry and targeted immune transcriptomics identify distinct profiles in patients with chronic myeloid leukemia receiving tyrosine kinase inhibitors with or without interferon-α

    doi: 10.1186/s12967-019-02194-x

    Figure Lengend Snippet: Regulatory T cells (Treg) in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. a Gating strategy used to identify blood Treg cells within the CD4 + and CD8 + T-cell compartment. b Percentage of CD4 + Treg cells in different patient groups (TKIs group, n = 33; TKIs plus IFN-α group, n = 8) and in healthy controls (n = 20). c Frequency of CD4 + Treg cells in patients with CML receiving imatinib (n = 26) or 2nd generation TKIs (n = 7). d Frequency of CD8 + Treg cells in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. The p values in the figure reflect statistically significant differences among study groups

    Article Snippet: Regulatory T cell (Treg) assessment Frozen PBMCs were thawed following the Cellular Technology Limited protocol (available online at http://www.immunospot.com ).

    Techniques:

    CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The PBMC and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, non-cryopreserved PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The PBMC and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, non-cryopreserved PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.

    Article Snippet: PBMC Donors with Low and High Responder Status to CEF Peptide Pool and Mumps Antigen Cryopreserved PBMC of twenty five donors were randomly selected from the ePBMC donor library provided by Cellular Technology Limited (CTL), Shaker Heights, OH, USA.

    Techniques: Ex Vivo, Standard Deviation, Isolation, Enzyme-linked Immunospot

    Comparison of mumps antigen elicited IFN-γ in freshly thawed and rested cryopreserved PBMC for low ( A ) and high responder subjects ( B ). Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or rested, the medium background was less than 10 spots per well. Non-parametric Wilcoxon signed-rank test was used to compare matched fresh vs . rested responses with a p -value ≤ 0.05 being considered significant.

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: Comparison of mumps antigen elicited IFN-γ in freshly thawed and rested cryopreserved PBMC for low ( A ) and high responder subjects ( B ). Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or rested, the medium background was less than 10 spots per well. Non-parametric Wilcoxon signed-rank test was used to compare matched fresh vs . rested responses with a p -value ≤ 0.05 being considered significant.

    Article Snippet: PBMC Donors with Low and High Responder Status to CEF Peptide Pool and Mumps Antigen Cryopreserved PBMC of twenty five donors were randomly selected from the ePBMC donor library provided by Cellular Technology Limited (CTL), Shaker Heights, OH, USA.

    Techniques:

    Comparison of CEF responses elicited by freshly thawed (“fresh”) and rested cryopreserved PBMC in ELISPOT assays. ( A ) CEF-specific responses among “low responders” who were defined as subjects who had fewer than 50 SFU in freshly thawed PMBC. ( B ) CEF-specific responses among “high responders” who were defined as subjects who had greater than 50 spots forming units (SFU) in freshly thawed PMBC. Data points obtained from the same donor before and after resting are connected by a line. Each data point represents the mean of triplicate antigen-stimulated wells. For each donor and antigen tested, the standard deviation was

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: Comparison of CEF responses elicited by freshly thawed (“fresh”) and rested cryopreserved PBMC in ELISPOT assays. ( A ) CEF-specific responses among “low responders” who were defined as subjects who had fewer than 50 SFU in freshly thawed PMBC. ( B ) CEF-specific responses among “high responders” who were defined as subjects who had greater than 50 spots forming units (SFU) in freshly thawed PMBC. Data points obtained from the same donor before and after resting are connected by a line. Each data point represents the mean of triplicate antigen-stimulated wells. For each donor and antigen tested, the standard deviation was

    Article Snippet: PBMC Donors with Low and High Responder Status to CEF Peptide Pool and Mumps Antigen Cryopreserved PBMC of twenty five donors were randomly selected from the ePBMC donor library provided by Cellular Technology Limited (CTL), Shaker Heights, OH, USA.

    Techniques: Enzyme-linked Immunospot, Standard Deviation