61-0289-41

antibody from Invitrogen Antibodies

Targeting: CD28

Provider product page for 61-0289-41

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [12]
Comments [0]
Validations
Flow cytometry [1]
Other assay [14]

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Product number: 61-0289-41 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD28 Monoclonal Antibody (CD28.2), PE-eFluor™ 610, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The CD28.2 monoclonal antibody reacts with the human CD28 molecule, a 44 kDa homodimer expressed by thymocytes, mature T cells and plasma cells. CD28 is a ligand for CD80 (B7-1) and CD86 (B7-2) and is a potent co-stimulator of T cells. Signaling through CD28 augments IL-2 and IL-2 receptor expression as well as cytotoxicity of CD3-activated T cells. Applications Reported: This CD28.2 antibody has been reported for use in flow cytometric analysis. Applications Tested: This CD28.2 antibody has been pre-titrated and tested by flow cytometric analysis of normal human peripheral blood cells. This can be used at 5 µL (0.25 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. PE-eFluor® 610 can be excited with laser lines from 488-561 nm and emits at 607 nm. We recommend using a 610/20 band pass filter (equivalent to PE-Texas Red®). Please make sure that your instrument is capable of detecting this fluorochome. Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 488-561 nm; Emission: 607 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: CD28.2
Vial size: 25 Tests
Concentration: 5 µL/Test
Storage: 4° C, store in dark, DO NOT FREEZE!

CD28 protein structure - 61-0289-41 shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 CD8 + T cells phenotype in CRC and OVC patients. A . An example of flow cytometric analysis of CD3 + CD8 + T cells analyzed for CD28 and Ki-67, upper panels, and HLA-DR and CD38, lower panels. Percentage of Ki-67 + CD8 T cells in CRC samples (n = 16) B . and OVC samples (n = 22) C . Co-expression of CD38 and HLA-DR on CD8 + T cells in CRC samples (D) and OVC samples (E) .

Supportive validation

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Experimental details: Figure 5 ALA-induced PpIX production and PDT of CD4 + CD25 + T cells. Healthy donor PBMCs were either activated in vitro with anti-CD3/IL-2 or anti-CD3/CD28 or kept non-activated (resting) for three days. ( A ) geometric mean fluorescence intensity (Geo. MFI) of PpIX in CD4 + CD25 + cells incubated with 3 mM ALA for 1 h at 37 degC after anti-CD3/IL-2 or anti-CD3/CD28 activation; ( B , C and D ) resting and activated cells were incubated with 3 mM ALA for 1 h and then exposed to the in-house built LED blue light as indicated. The survivals of CD4 + CD25 + T cells were measured before light or 20 h after light exposure with flow cytometry as described in Figure 3 ; ( B ) for resting cells; while ( C and D ) for activated cells with anti-CD3/IL-2 and anti-CD3/CD28, respectively. * p < 0.05.

Supportive validation

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Experimental details: FIGURE 1 Identification of Tscm CD4 + CD45RA + CD45RO - CD62L + CCR7 + CD127 + CD27 + CD28 + CD95 + CD122 + T (Tscm) cell in human blood and lymph nodes. PBMCs were isolated from the blood of non-small cell lung cancer (NSCLC) patients (n=15) (NSCLC-PBMC) and healthy donors (n=11) (HD-PBMC); lymphocytes were isolated from the tumor-infiltrated lymph node of NSCLC patients who were collected blood at same time (n=7) (NSCLC-Ly); lymphocytes were isolated from the healthy lymph node of non lung cancer patients (n=7) (Normal-Ly), analyzed by flow cytometry. A, Representative flow cytometric analyses of CD4 + CD45RA + CD45RO - CD62L + CCR7 + CD127 + CD27 + CD28 + CD95 + CD122 + T cells, indicating Tscm cells. B, The frequency of the CD4 + Tscm cells in the HD-PBMC, NSCLC-PBMC, Normal-Ly, NSCLC-Ly. The events of CD4 + Tscm cells in the blood and lymph node from NSCLC patients and healthy donors, expressed as the mean+-SEM. C, The frequency of the CD8 + Tscm cells in the HD-PBMC, NSCLC-PBMC, Normal-Ly, NSCLC-Ly, expressed as the mean+-SEM. D, The events of Tscm of CD4 + and CD8 + cells in the blood and the lymph node from NSCLC patients and healthy donors. HD indicates healthy donors; IFN, interferon; PBMC, peripheral blood mononuclear cells; Tscm cell, stem cell-like memory T cell. * P

Supportive validation

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Experimental details: Figure 3. Altered FoxO1 gene expression in CD8 + CD28 - T cells. (A) Microarray analysis in CD8 + CD28 + and CD28 - T cells, predicting FoxO1 as an altered transcriptional regulator in CD8 + CD28 - T cells. (B) CCR7 , CD62L , IL7R , and KLRG1 mRNA were assessed by qRT-PCR and normalized to RPL13A mRNA from sorted human T cell populations ( n = 5, paired one-way ANOVA). (C) Peripheral blood mononuclear cells were stained for CD3, CD8, CD28, and indicated markers as in B and analyzed by flow cytometry ( n = 5, paired one-way ANOVA). (D and E) FoxO1 expression in sorted human T cells was measured by Western blot (representative, n = 7, paired two-tailed Student's t test). (F) FoxO1 mRNA was analyzed by qRT-PCR and normalized to RPL13A ( n = 8). (G and H) CD28 + and CD28 - T cells were treated with 20 uM MG132 for 6 h, and FoxO1 expression was measured by Western blot ( n = 3, unpaired two-tailed Student's t test, G shows CD28 - T cells). (I and J) SIRT1 knockdown by Cas9-RNP nucleofection and Western blot for SIRT1 and FoxO1 expression ( n = 9, two-way ANOVA). (K) Nucleofection of recombinant SIRT1 protein into CD8 + T cells and Western blot for SIRT1 and FoxO1 protein 18 h after nucleofection (representative, n = 2). (L) Densitometry of two independent experiments ( n = 2). Data are mean +- SEM of individual donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.000. ns, not significant.

Supportive validation

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Experimental details: Figure 2 Confirmation of CD19 CAR plasmid into the transfected producer cell line. ( A ) Agarose gel demonstrating cellular mRNA expression (eGFP, CD8a, and CD28) in CD19 CAR plasmid transfected HEK293T cells and control non-transfected cells (WT-control). The beta-actin band is same for each gene of interest because same sample/cDNA was used for PCR amplification of eGFP, CD8a, and CD28. ( B ) Protein expression by flow cytometry (contour plot) demonstrating surface protein expression of CD3z, CD8a, and CD28 co-localized with eGFP expression in CD19 CAR plasmid transfected HEK293T cells (CD19 CAR) and non-transfected cells (WT-control). Bar graph is representation of the three replicates. p -value (*** p < 0.001). This is a representation of cellular CD3z, CD8a, and CD28 expression.

Supportive validation

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Experimental details: Figure 5. SIRT1 undergoes lysosomal degradation during aging in mouse and human. a,b, Spleens ( a ) and testes ( b ) from young (2-4 months) and aged (19-26 months) C57BL/6 mice were analyzed by western blotting and RT-qPCR. Data are mean +- s.e.m. ; unpaired two-tailed Students' t-test; n = 4 animals. c,d, Spleens ( c ) and testes ( d ) were analyzed by western blotting and RT-qPCR for SIRT1 expression, from aged (19-24 months) mice subjected to daily i.p. injection of 10 mg/kg Lys05 in PBS or PBS control in 100 muL volume for two weeks. Data are mean +- s.e.m. ; two-tailed Mann-Whitney test. For spleen protein, control group n = 8 animals, Lys05 group n = 7 animals; RNA, control group n = 8 animals, Lys05 group n = 6 animals. For testis protein and RNA, control group n=6 animals, Lys05 group n=6 animals. e. HSPC populations were isolated from young (2-4 months) and aged (20-26 months) C57BL/6 mice, cultured with or without 2 muM Lys05 for 24 hours and analyzed by western blotting and RT-qPCR. For protein, data are mean +- s.e.m. ; one-way ANOVA coupled with Tukey's test; n=6 independent experiments. For RNA, data are mean +- s.e.m. ; unpaired two-tailed Students' t-test; n=4 independent experiments. f. Freshly sorted CD8 + CD28 + (control) and CD8 + CD28 - T cells were treated with Lys05 at doses of 0 and 5 muM for 14 hours, and then were harvested and analyzed by western blotting. Donor age: 53, 54 and 66. Data are mean +- s.d. ; unpaired two-tailed Students' t-test; n = 3

Supportive validation

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Experimental details: Fig. 1 Characterization of CD26 expression, mDPP4 enzymatic activity, and cytokine expression in T helper cell lines. (A, B) Jurkat E6 and H9 Th1 cells were harvested and analyzed for the expression of T cell-specific surface markers, including CD3, CD4, and CD26 (A), and for their mDPP4 enzymatic activity (B). (C) H9 Th1 cells were treated with various T cell activators, including T-Activator CD3/CD28, PWM (10 ug/mL), and PMA (10 ug/mL) for 3 h. The expressions of 18 cytokines and 6 MMPs were measured in the culture supernatants of treated cells using two different panels of fluorescent multiplex bead assays. Activated T helper cell-specific cytokines, including IL-2, IL-10, IL-13, GM-CSF, IFN-gamma, and TNF-alpha, are shown. VEGF and MMP 9 are also included as detected high in all the samples. (D-F) H9 Th1 cells were treated with 10 ug/mL PWM for 12 h and subjected to mDPP4 enzymatic assays (D), FACS analysis for T helper cell-specific surface markers such as CD3, CD4, CD26, and CD28 (E), and analysis for apoptosis using an annexin V kit and flow cytometry (F). All analyses were repeated in three independent batches, and the data are shown as the means and standard deviation. ** p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Expression pattern of CD27 and CD28 on gammadelta T cells in AR. (A) Representative flow cytometry plots of the expression of CD27 and CD28 on gammadelta T cells among PBMCs from HC (n=12) and AR (n=14) subjects. (B-G) Quantification of the percentage of (B) CD27+, (C) CD28+, (D) CD27 + CD28 + , (E) CD27 + CD28 - , (F) CD27 - CD28 + and (G) CD27 - CD28 - gammadelta + T cells in PBMCs. Values are expressed as the mean +- standard deviation. AR, allergic rhinitis; HC, healthy controls; PBMCs, peripheral blood mononuclear cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Aging and replicative senescence markers in human T lymphocytes. PBMCs from healthy young (< 28 y) and old (> 56 y) donors were stained for CD28 and CD57 and run on LSR II flow cytometer. (A) Autophagy levels (Mean BDS) in CD8 + T cells from young (< 28 y, n = 8) and old (> 56 y, n = 8) donors under basal and basal+I (for 2 h) conditions (mean +- SEM, *p < 0.0499 between young and old basal+I). (B) Representative dot plots from a young and an old donor showing percentages of CD28 and CD57 cells gated on CD8 + T cells. (C) Bar graph showing % of CD8 + lymphocytes with CD28 and CD57 markers in four young and old donors (mean +- SEM, p = 0.0571 for CD8 CD28 population and *p = 0.0286 for CD8 CD57 population). (D) Overlaid histogram of gammaH2AX (DNA double-strand break) levels of CD8 + lymphocytes from three young and old donors gated on CD28 + CD57 - population (geometric mean +- SEM, *p = 0.0286). (E) Overlaid histogram of FAS (CD95) levels of CD8 + lymphocytes from four young and old donors gated on CD28 + CD57 - population (geometric mean +- SEM, *p = 0.0286). PBMCs from four healthy young and old donors were cultured under control and starved conditions for 2 h and stained for CD8, CD57, LC3 and Lyso-ID. (F) Colocalization of LC3 and lysosomal marker in CD8 + CD57 +/- cells, expressed as mean BDS ratio between starved and basal treatments (mean +- SEM, n = 5 (young donors), n = 8 (old donors), **p = 0.0049, *p = 0.035).