Freshly isolated PBMCs were cultured with PHA, Con A, LPS, and, PWM each at 1 μg/mL final concentration as stimulators to determine the numbers of IFN-γ– or perforin-producing cells. To enumerate and quantify IFN-γ or perforin-producing cells, we followed the standard enzyme-linked immunospot (ELISPOT) protocol, as reported earlier [13 (link), 18 –20 (link)]. Briefly, aliquots of PBMCs (105/well) were seeded in duplicate wells of 96-well plates (polyvinylidene difluoride–backed plates, MAIP S 45, Millipore, Bedford, MA), pre-coated with primary IFN-γ or perforin antibody, and were stimulated with the various mitogens. After incubation for 30–32 h at 37°C, the cells were removed, the wells were thoroughly washed with 1× PBS, and spots were developed as per the manufacturer’s direction. Purple-colored spots representing individual cells secreting IFN-γ or perforin were counted by an independent agency (ZellNet Consulting, Fort Lee, NJ) by using the KS-ELISPOT automatic system (Carl Zeiss, Inc. Thornwood, NY) for quantitative analysis of the number of IFN-γ or perforin spot-forming cells for 105 input PBMCs. Responses were considered positive when the number of spot-forming cells with the test mitogens was at least five spots above the background control values from cells cultured in the medium alone. Data were represented as SFCs per 105 PBMCs.
Maip s 45
Manufactured by Merck Group
Sourced in United States
MAIP S 45 is a laboratory equipment product from Merck Group. It is a centrifuge designed for separating materials of different densities in liquid samples. The device operates at a maximum speed of 4,500 rpm and has a maximum capacity of 45 ml per sample.
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Lab products found in correlation
9 protocols using maip s 45
1
Quantifying IFN-γ and Perforin-Producing Cells
2
Quantification of HIV-Specific CD4 T-Cell Responses
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Freshly isolated CD8 depleted PBMC were seeded in 96-well polyvinylidene difluoride-backed plates (MAIP S45, Millipore) that had been coated with an anti-IFN-γ MAb 1-D1k (0.5 µg/ml, Mabtech) overnight at 4 °C.
HIV-specific CD4 T-cell responses were quantified, as described previously [37 (link)]. Overlapping peptides were added to 1 × 105 CD8 depleted PBMC per well at a final concentration of 12.5 µg/ml. The plates were then incubated for a 14–16 hours at 37°C and 5% CO2. IFN-γ positive were detected using a biotinylated secondary anti-IFN-γ MAb 7-B6-1 (0.5 µg/ml, Mabtech), a streptavidin-alkaline phosphatase conjugate (Mabtech) and TMB substrate (Mabtech).
IFN-γ-producing cells were counted and expressed as spot-forming cells (SFC) per 106 PBMC. Negative controls were always < 10 SFC per 106 cells. As positive controls, we incubated PBMC with phytohemagglutinin or anti-CD3/CD28 coated beads. Wells were considered positive if they had at least 20 SFC/106 PBMC.
HIV-specific CD4 T-cell responses were quantified, as described previously [37 (link)]. Overlapping peptides were added to 1 × 105 CD8 depleted PBMC per well at a final concentration of 12.5 µg/ml. The plates were then incubated for a 14–16 hours at 37°C and 5% CO2. IFN-γ positive were detected using a biotinylated secondary anti-IFN-γ MAb 7-B6-1 (0.5 µg/ml, Mabtech), a streptavidin-alkaline phosphatase conjugate (Mabtech) and TMB substrate (Mabtech).
IFN-γ-producing cells were counted and expressed as spot-forming cells (SFC) per 106 PBMC. Negative controls were always < 10 SFC per 106 cells. As positive controls, we incubated PBMC with phytohemagglutinin or anti-CD3/CD28 coated beads. Wells were considered positive if they had at least 20 SFC/106 PBMC.
3
Quantification of Antigen-Specific IFNγ
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Five weeks after the last immunization, the levels of antigen-specific IFNγ in spleen and popliteal lymph nodes were analyzed. IFNγ was tested using an enzyme-linked immunospot (ELISPOT) assay following a 20-hour incubation of cells with specific antigens including DBD-Ag85a (10 μg/ml), DBD-ESAT6-CFP10 (10 μg/ml) and DBD (10 μg/ml). Binding IFNγ antibody (100 μL; 4 μg/ml in sterile PBS; clone R4-6A2, BD PharMingen) were incubated for 18–20 hours at +4°C in 96 well plates (Millipore, MAIP S45, USA). The plates were treated with 200 μL/well RPMI supplemented by 5% FCS at 37°C for 2–4 hours. Later, the cells in doublets (0.5–8×105/well in 200 μL RPMI supplemented with 5% FCS; 5% CO2) with antigen (10 μg/ml) were incubated for 48 hours at 37°C. The plates were washed 5 times with PBS (200 μL/well) and once with distilled water and incubated with detection antibodies (1 μg/ml in PBS; clone XMG1.2, BD PharMingen) for 2–4 hours. After washing (6 washes in 200 μL/well PBS) and incubation with alkaline phosphatase conjugate (1:1000 dilution, Sigma; 1–2 hours at RT), colorimetric substrate (Sigma, in PBS) was used for visualization of separate dots. ELISPOT reader (BioReader 4000 Pro-X, BIO-SYS, Germany) was used to calculate the number of dots.
4
Evaluating SIVmac239 Gag-specific T-cell Responses
5
Quantifying IFN-γ-producing cells by ELISPOT
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Freshly-isolated PBMCs as described above, were stimulated with the mitogens PHA, Con A, LPS and PWM (each at 2 μg/mL final concentration) to determine the numbers of IFN-γ-producing cells by the Enzyme Linked Immuno Spot (ELISPOT) assay using the methodology reported previously [19 (link), 23 (link), 24 (link)]. Briefly, aliquots of PBMCs (105/well) were seeded in duplicate wells of 96-well plates (polyvinylidene difluoride backed plates, MAIP S 45, Millipore, Bedford, MA) pre-coated with the primary IFN-γ antibody and the lymphocytes were stimulated with the different mitogens. After incubation for 18–20 hr. at 37°C, the cells were removed and the wells were thoroughly washed with PBS and developed as per protocol provided by the manufacturer. Blue-Purple colored spots representing individual cells secreting IFN-γ were counted by an independent agency (Zellnet Consulting, New Jersey, NJ) using the KS-ELISPOT automatic system (Carl Zeiss, Inc. Thornwood, NY) for the quantitative analysis of the number of IFN-γ spot forming cells (SFC) for 105 input of PBMCs. Responses were considered positive when the numbers of SFC with the test antigen were at least five spots above the background control values from cells cultured in the media alone.
6
Tick-Borne Encephalitis Virus Antibody and Cellular Immune Responses
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TBEv‐specific antibody titers were measured before (week 0), during (week 8), and after (week 28) the TBEv vaccination course by ELISA and TBEv‐neutralization according to published protocols (Stiasny et al., 2012 ). The TBEv‐specific cellular immune response was assessed at weeks 0 and 26 by IFN‐γ enzyme‐linked immunosorbent spot (ELISpot) assay using pools of overlapping peptides for all structural proteins of TBEv. Briefly, 2 × 105 thawed PBMCs/well from the same donor at week 0 and week 26 were stimulated in α‐IFN‐γ (clone 1‐D1K; Mabtech, Nacka Strand, Sweden)‐coated 96‐well ELISpot plates (MAIP S45; Millipore) for 18 h with 2 × 104 freshly generated autologous monocyte‐derived DCs. For antigen‐specific stimulation, five pools of overlapping peptides encompassing all structural proteins of TBEv were used at a final concentration of 1μg mL−1 (15‐mer peptides overlapping by 5 amino acids; BMC Microcollections, Germany). Washed plates were then incubated with α‐IFN‐γ‐biotin (7‐B6‐1; Mabtech) followed by streptavidin–alkaline phosphatase (Mabtech), developed with color reagents (170‐6432; Bio‐Rad , Marnes‐la‐Coquette, France) and analyzed in an automated ELISpot reader (AID). The number of spot‐forming units (SFU) was calculated after subtraction of the unstimulated control.
7
PBMC Stimulation and ELISPOT Assay
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Freshly-isolated PBMC, as described above, were stimulated with the mitogens CpG ODN (5 μg/ml and 50 μg/ml final concentrations) and 5 μg/ml of Con A to determine the numbers of IFNγ- or IL12p40-producing cells by the Enzyme Linked Immuno Spot (ELISPOT) assay using the methodology reported earlier (Nehete et al., 2003 (link), 2005 (link)). Briefly, aliquots of PBMC (105/well) were seeded in triplicate wells of 96-well plates (polyvinylidene difluoride backed plates, MAIP S 45, Millipore, Bedford, MA, USA) pre-coated with the primary IFNγ or IL12p40 antibody, and the lymphocyte were stimulated with the different mitogens or antigen. After incubation for 30–32 h at 37°C, the cells were removed, and the wells were thoroughly washed with PBS and developed as per the protocol provided by the manufacturer. Purple colored spots representing individual cells secreting IFNγ or IL12p40 were counted by an independent agency (Zellnet Consulting, New Jersey, NJ, USA) using the KS-ELISPOT automatic system (Carl Zeiss, Inc. Thornwood, NY, USA) for the quantitative analysis of the number of IFNγ or IL12p40 spot forming cells (SFC) for 105 input PBMC. Responses were considered positive when the number of SFCs with the test antigen was at least five and was five above the background control values from cells cultured in the medium alone.
8
ELISPOT Quantification of IFN-γ Producing PBMCs
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Freshly-isolated PBMCs, as described above, were stimulated with the mitogens PHA, Con A, and PWM (each at 5 μg/mL final concentration) to determine the numbers of IFN-γ-producing cells by the Enzyme Linked Immuno Spot (ELISPOT) assay using the methodology reported earlier [20 (link), 22 (link)]. Briefly, aliquots of PBMCs (105/well) were seeded in triplicate wells of 96-well plates (polyvinylidene difluoride backed plates, MAIP S 45, Millipore, Bedford, MA) precoated with the primary IFN-γ antibody and the lymphocytes were stimulated with the different mitogens. After incubation for 30 hr at 37°C, the cells were removed and the wells were thoroughly washed with PBS and developed as per protocol provided by the manufacturer. Purple colored spots representing individual cells secreting IFN-γ were counted by an independent agency (Zellnet Consulting, New Jersey, NJ) using the KS-ELISPOT automatic system (Carl Zeiss Inc., Thornwood, NY) for the quantitative analysis of the number of IFN-γ spot forming cells (SFC) for 105 input PBMCs. Responses were considered positive when the numbers of SFC with the test antigen were at least five and also were five above the background control values from cells cultured in the medium alone.
9
Quantifying HIV-specific CD4 T-cell Responses
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Freshly isolated CD8 depleted PBMC were seeded in 96-well polyvinylidene di uoride-backed plates (MAIP S45, Millipore) that had been coated with an anti-IFN-g MAb 1-D1k (0.5 µg/ml, Mabtech) overnight at 4°C. HIV-speci c CD4 T-cell responses were quanti ed, as described previously [37] . Overlapping peptides were 1 x 10 5 CD8 depleted PBMC per well at a nal concentration of 12.5 µg/ml. The plates were then incubated for a 14-16 hours at 37°C and 5% CO 2 . IFN-g positive were detected using a biotinylated secondary anti-IFN-g MAb 7-B6-1 (0.5 µg/ml, Mabtech), a streptavidin-alkaline phosphatase conjugate (Mabtech) and TMB substrate (Mabtech). IFN-g-producing cells were counted and expressed as spot-forming cells (SFC) per 10 6 PBMC. Negative controls were always < 10 SFC per 10 6 cells. As positive controls, we incubated PBMC with phytohemagglutinin or anti-CD3/CD28 coated beads. Wells were considered positive if they had at least 20 SFC/10 6 PBMC.
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