Patent Application
20230324395
The present application claims priority from Chinese Patent Application No. 202210375754.X filed on Apr. 11, 2022, the contents of which are incorporated herein by reference in their entirety.
The present disclosure relates to the technical field of mass cytometry, in particular to an antibody combination for substituting a side scatter signal in mass cytometry hematologic tumor immunophenotyping and use thereof.
To select a therapeutic regimen correctly, the precondition is an accurate classification of hematologic tumors. Currently, the mode that is commonly used in the world is cell morphology, immunology, cytogenetics and molecular biology classification, i.e., MICM classification. Among them, multi-parameter flow cytometry of immunology classification plays an important role, which improves the identification accuracy of specific disease types based on the immune signature of patient tumor cells.
As for the multi-parameter flow cytometry, CD molecules, such as stem/progenitor cell antigens, bone marrow cell line associated antigens, red blood cells, B cells, T cells, NK cells, megakaryocytes and other associated antigens, on the surface of bone marrow cells are detected by fluorescent antibodies. Common antibody combinations are usually three- or four-color schemes, using three or four fluoresceins to label antibodies separately. Flow cytometry is usually carried out for hematologic tumor classification by gating. That is, cell subsets are distinguished deeply step by step. For example, in T cells, the T cells are distinguished using CD3, and then CD3+CD4+T cells and CD3+CD8+T cells are distinguished using CD4 and CD8. The side scatter (SSC) signal of flow cytometers also plays an important role. At present, for the analysis of flow cytometry detection results, the first-stage gating strategy is to use CD45 and SSC as horizontal and vertical coordinates respectively to distinguish CD45 negative nucleated red blood cell subsets, CD45dimSSC-low primitive and juvenile cell subsets, CD45dimSSC-high mature granulocyte subsets, CD45+SSC-low lymphocyte subsets, and CD45+SSC-intermediate monocyte subsets, and further analyze each subset by different antibodies.
For completing hematologic tumor classification, it usually needs to detect 30 or more CD molecule antibodies and some other classification antibodies. Since commonly used flow cytometers in clinic is of 4-6 color, i.e., it can detect 4-6 proteins on a cell at a time. To complete the detection of 30 or more antibodies, a tube of bone marrow needs to be divided into 8-10 tubes of samples for staining and analysis respectively (some antibodies require repeated detection to determine cell subsets), resulting in large sample sizes. The process is cumbersome and it is not possible to carry out simultaneous analysis of 30 or more protein parameters for a single cell. The multi-parameter flow cytometry is also interfered by background fluorescence of samples. The emission wavelengths of different fluoresceins are overlapped. Therefore, even light filters are used, compensation regulation is still required.
Mass cytometry is a new multi-parameter flow cytometry that uses metal-labeled antibodies with extremely low abundance in organisms such as rare earth metals, and uses time-of-flight mass spectrometry to accurately detect the metal content in each cell. Due to the detection characteristics of mass spectrometers, there is almost no interference between different metal signals and no compensation regulation is required. It is possible to simultaneously detect 43 antibodies (including CD molecules) on a single cell with single-tube detection, which has a methodological advantage for cell classification of complex cell types, overcomes the defects of the current 4-6 color traditional flow cytometry, and has the potential to be used as a hematologic tumor immunodetection platform.
For detection of hematologic tumors, the traditional flow cytometry uses CD45 and SSC for gating, which can quickly distinguish nucleated red cell subsets, primitive and juvenile cell subsets, monocyte subsets, lymphocyte subsets, mature granulocyte subsets, and the like. Since mass cytometry uses mass spectrometry methodology, in which cells are completely ionized, SSC of traditional flow cytometry is not included in the detection process. When applied to hematologic tumors, the first-stage gating similar to flow cytometry, namely CD45 and SSC combined gating, cannot be carried out, and the major cell subsets cannot be distinguished. Therefore, the clinical application and scientific research of mass cytometry in the hematologic tumor field are limited. Thus, it is necessary to develop an antibody combination that can substitute the traditional SSC to make up for the deficiency of mass cytometry and bring its multi-parameter synchronous detection into full play, such that mass cytometry can be applied in the immunophenotype detection of hematologic tumors better.
The present disclosure aims to provide an antibody combination for substituting a side scatter signal in mass cytometry hematologic tumor immunophenotyping and use thereof to solve the defects in the prior art.
The present disclosure adopts the following technical solutions:
The first aspect of the present disclosure provides an antibody combination for substituting a side scatter signal in mass cytometry hematologic tumor immunophenotyping, including a Lactoferrin antibody and a Lysozyme antibody, the Lactoferrin antibody and the Lysozyme antibody having metal tags respectively, and the metal tags of the Lactoferrin antibody and the Lysozyme antibody being different.
Further, the metal tag is selected from 89Y, 115In, 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 157Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, 176Yb, 195Pt, 197Au, 198Pt, and 209Bi.
The second aspect of the present disclosure provides use of the antibody combination above in mass cytometry hematologic tumor immunophenotyping.
Further, the following steps are included:
Furthermore, the metal tag is selected from 89Y, 115In, 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 157Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, 176Yb, 195Pt, 197Au, 198Pt, and 209Bi.
The third aspect of the present disclosure provides a gating method for mass cytometry hematologic tumor immunophenotyping, including the following steps:
Further, the metal tag is selected from 89Y, 115In, 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 157Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, 176Yb, 195Pt, 197Au, 198Pt, and 209Bi.
The fourth aspect of the present disclosure provides a kit for mass cytometry hematologic tumor immunophenotyping, consisting of 43 monoclonal antibodies with metal tags, as shown in the following table:
where numbers 1, 3, 12, 14, 17, 23, 26, 34, and 38 are intracellular antibodies, and others are extracellular antibodies.
The fifth aspect of the present disclosure provides use of the kit above in mass cytometry hematologic tumor immunophenotyping.
Further, the following steps are included:
1. The present disclosure provides the antibody combination for substituting a side scatter signal in mass cytometry hematologic tumor immunophenotyping. The antibody combination consisting of the Lactoferrin antibody and the Lysozyme antibody substitutes the traditional flow cytometry side scatter signal, such that the function of a traditional flow cytometer to detect SSC is realized in the mass cytometer. The antibody combination is applied, in combination with the CD45 antibody, in mass cytometry hematologic tumor immunophenotyping, which can realize the effect of traditional flow cytometry SSC and CD45 two-dimensional plotting. Moreover, combined with other common antibodies for hematologic tumor immunophenotyping, hematologic tumor immunophenotyping can be carried out, the bone marrow cells are divided into large groups and distinguished, and abnormal subsets can be found.
2. The present disclosure provides the gating method for mass cytometry hematologic tumor immunophenotyping. The mature granulocyte subset, the monocyte subset, and other cell subsets are distinguished first using the Lactoferrin antibody and the Lysozyme antibody. Then the other cell subsets are grouped by the CD45 antibody into the primitive and juvenile cell or/abnormal cell subsets, the nucleated red blood cell subset, and the lymphocyte subset. Then the expression of antigens of related subsets is analyzed by other common antibodies for hematologic tumor immunophenotyping to determine whether there is abnormal expression of the antigens of related subsets, realizing mass cytometry hematologic tumor immunophenotyping. According to the present disclosure, the Lactoferrin antibody and the Lysozyme antibody are used for the first time, are combined with a CD45 antibody for two-stage gating strategy, and are combined with a mass cytometer to substitute traditional flow CD45/SSC to distinguish mature granulocytes, monocytes, nucleated red blood cells, lymphocytes, primitive and juvenile cells, and abnormal cell subsets in bone marrow. This overcomes the technical difficulty that the mass cytometry cannot detect SSC in hematologic tumor cell analysis. Combined with the multi-parameter high-throughput characteristics of the mass cytometry, the present disclosure can improve the depth of present hematologic tumor immunophenotyping, and is convenient for clinicians to analyze hematologic tumors according to a traditional flow cytometry mode.
3. The present disclosure provides the kit for mass cytometry hematologic tumor immunophenotyping, consisting of 43 monoclonal antibodies with metal tags. The kit of the present disclosure overcomes the technical difficulty that mass cytometry cannot detect SSC in hematologic tumor cell analysis, realizes the accurate classification of hematologic tumor cells by mass cytometry, and can detect 43 protein markers simultaneously on a single hematologic tumor cell, increasing the sensitivity, accuracy and economy of detection. By testing, the kit of the present disclosure can realize, by just single-tube detection, the effect of the traditional flow cytometer that requires 8-10 tubes for detection, and expands the range and ability of hematologic tumor-related immunophenotype analysis, without single stain control of each channel, without regulating fluorescence compensation, and reduces experimental procedures and sample sizes, laying a foundation for further realization of intelligence and automation of hematologic tumor immunophenotyping. With the aid of the mass cytometer, by using the kit of the present disclosure, the type and nature of hematologic tumor cells can be rapidly and accurately analyzed and the level of positive cells can be determined, which has important guiding significance for prognosis and formulation of clinical therapeutic regimen. Moreover, the detection samples are saved, and more markers can be detected for a single cell at the same time, which also provides more abundant data for the research of hematologic tumors.
4. With the antibody combination, the gating method and the kit of the present disclosure, it is conducive to the use of the mass cytometer to the standardization, normalization, automation and intelligence of the hematologic tumor immunophenotyping.
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The present disclosure will be further explained below in conjunction with the examples and drawings. The following examples are only used to illustrate the present disclosure, but cannot be used to limit the implementation scope of the present disclosure.
The antibodies involved in the following examples are as shown in Table 1:
where cCD3, cIgM, MPG, λ, Lactoferrin, κ, Lysozyme, CD79a, and TdT antibodies with numbers 1, 3, 12, 14, 17, 23, 26, 34, and 38 are intracellular antibodies, and others are extracellular antibodies.
1) Fresh bone marrow of healthy human was prepared, with mature red blood cells removed.
2) 1-3×10{circumflex over ( )}6 cells were taken and re-suspended with PBS, the volume was adjusted to 1 mL, 50 μL−1 mL of 194Pt (0.1-1 μM) was added, and staining was carried out at room temperature for 2 min to determine whether the cells were dead or alive.
3) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, and 50 μL of blocking buffers was added for blocking on ice for 20 min. The blocking buffer consisted of 0.5 μL of human immunoglobulin solutions (including 15-25 parts by mass of human immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of mouse immunoglobulin solutions (including 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of rat immunoglobulin solutions (including 15-25 parts by mass of rat immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of hamster immunoglobulin solutions (including 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), and 48 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers).
4) 50 μL of extracellular antibody mixed liquid (0.5 μL of each of 34 extracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 33 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added, cells were resuspended, and staining was carried out on ice for 30 min.
5) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, 1 mL of fixation/permeabilization solutions containing 0.5 v/v % c single-cell indicator 191/193 Ir was added and cells were re-suspended overnight at 4° C.
6) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction, for the control group, 50 μL of fixation-permeabilization solutions was added as blank control, for the experimental group, 50 μL of intracellular antibody mixed liquid (0.5 μL of each of 9 intracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 45.5 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers)) was added, cells were suspended and placed on ice for 30 min.
7) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
8) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
9) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
10) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
11) The sample was filtered, the cells were counted, the volume was adjusted, and preparation was carried out for on-machine mass cytometry detection.
The analysis results are shown in
1) Fresh bone marrow of patients with acute lymphoblastic leukemia was prepared, with mature red blood cells removed.
2) 1-3×10{circumflex over ( )}6 cells were taken and re-suspended with PBS, the volume was adjusted to 1 mL, 50 μL−1 mL of 194Pt (0.1-1 μM) was added, and staining was carried out at room temperature for 2 min to determine whether the cells were dead or alive.
3) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, and 50 μL of blocking buffers was added for blocking on ice for 20 min. The blocking buffer consisted of 0.5 μL of human immunoglobulin solutions (including 15-25 parts by mass of human immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of mouse immunoglobulin solutions (including 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of rat immunoglobulin solutions (including 15-25 parts by mass of rat immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of hamster immunoglobulin solutions (including 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), and 48 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers).
4) 50 μL of extracellular antibody mixed liquid (0.5 μL of each of 34 extracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 33 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added, cells were resuspended, and staining was carried out on ice for 30 min.
5) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, 1 mL of fixation/permeabilization solutions containing 0.5 v/v % c single-cell indicator 191/193 Ir was added and cells were re-suspended overnight at 4° C.
6) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction, for the control group, 50 μL of fixation-permeabilization solutions was added as blank control, for the experimental group, 50 μL of intracellular antibody mixed liquid (0.5 μL of each of 9 intracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 45.5 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers)) was added, cells were suspended and placed on ice for 30 min.
7) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
8) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
9) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
10) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
11) The sample was filtered, the cells were counted, the volume was adjusted, and preparation was carried out for on-machine mass cytometry detection.
The analysis results are shown in
1) Fresh bone marrow of patients with acute myelogenous leukemia was prepared, with mature red blood cells removed.
2) 1-3×10{circumflex over ( )}6 cells were taken and re-suspended with PBS, the volume was adjusted to 1 mL, 50 μL−1 mL of 194Pt (0.1-1 μM) was added, and staining was carried out at room temperature for 2 min to determine whether the cells were dead or alive.
3) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, and 50 μL of blocking buffers was added for blocking on ice for 20 min. The blocking buffer consisted of 0.5 μL of human immunoglobulin solutions (including 15-25 parts by mass of human immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of mouse immunoglobulin solutions (including 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of rat immunoglobulin solutions (including 15-25 parts by mass of rat immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of hamster immunoglobulin solutions (including 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), and 48 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers).
4) 50 μL of extracellular antibody mixed liquid (0.5 μL of each of 34 extracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 33 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added, cells were resuspended, and staining was carried out on ice for 30 min.
5) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, 1 mL of fixation/permeabilization solutions containing 0.5 v/v % c single-cell indicator 191/193 Ir was added and cells were re-suspended overnight at 4° C.
6) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction, for the control group, 50 μL of fixation-permeabilization solutions was added as blank control, for the experimental group, 50 μL of intracellular antibody mixed liquid (0.5 μL of each of 9 intracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 45.5 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers)) was added, cells were suspended and placed on ice for 30 min.
7) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
8) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
9) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
10) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
11) The sample was filtered, the cells were counted, the volume was adjusted, and preparation was carried out for on-machine mass cytometry detection.
The analysis results are shown in
1) Fresh bone marrow of patients with myelodysplastic syndrome was prepared, with mature red blood cells removed.
2) 1-3×10{circumflex over ( )}6 cells were taken and re-suspended with PBS, the volume was adjusted to 1 mL, 50 μL−1 mL of 194Pt (0.1-1 μM) was added, and staining was carried out at room temperature for 2 min to determine whether the cells were dead or alive.
3) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, and 50 μL of blocking buffers was added for blocking on ice for 20 min. The blocking buffer consisted of 0.5 μL of human immunoglobulin solutions (including 15-25 parts by mass of human immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of mouse immunoglobulin solutions (including 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of rat immunoglobulin solutions (including 15-25 parts by mass of rat immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of hamster immunoglobulin solutions (including 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), and 48 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers).
4) 50 μL of extracellular antibody mixed liquid (0.5 μL of each of 34 extracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 33 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added, cells were resuspended, and staining was carried out on ice for 30 min.
5) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, 1 mL of fixation/permeabilization solutions containing 0.5 v/v % c single-cell indicator 191/193 Ir was added and cells were re-suspended overnight at 4° C.
6) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction, for the control group, 50 μL of fixation-permeabilization solutions was added as blank control, for the experimental group, 50 μL of intracellular antibody mixed liquid (0.5 μL of each of 9 intracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 45.5 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers)) was added, cells were suspended and placed on ice for 30 min.
7) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
8) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
9) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
10) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
11) The sample was filtered, the cells were counted, the volume was adjusted, and preparation was carried out for on-machine mass cytometry detection.
The analysis results are shown in
1) Fresh bone marrow of patients with multiple myeloma was prepared, with mature red blood cells removed.
2) 1-3×10{circumflex over ( )}6 cells were taken and re-suspended with PBS, the volume was adjusted to 1 mL, 50 μL−1 mL of 194Pt (0.1-1 μM) was added, and staining was carried out at room temperature for 2 min to determine whether the cells were dead or alive.
3) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, and 50 μL of blocking buffers was added for blocking on ice for 20 min. The blocking buffer consisted of 0.5 μL of human immunoglobulin solutions (including 15-25 parts by mass of human immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of mouse immunoglobulin solutions (including 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of rat immunoglobulin solutions (including 15-25 parts by mass of rat immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of hamster immunoglobulin solutions (including 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), and 48 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers).
4) 50 μL of extracellular antibody mixed liquid (0.5 μL of each of 34 extracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 33 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added, cells were resuspended, and staining was carried out on ice for 30 min.
5) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, 1 mL of fixation/permeabilization solutions containing 0.5 v/v % c single-cell indicator 191/193 Ir was added and cells were re-suspended overnight at 4° C.
6) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction, for the control group, 50 μL of fixation-permeabilization solutions was added as blank control, for the experimental group, 50 μL of intracellular antibody mixed liquid (0.5 μL of each of 9 intracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 45.5 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers)) was added, cells were suspended and placed on ice for 30 min.
7) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
8) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
9) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
10) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.
11) The sample was filtered, the cells were counted, the volume was adjusted, and preparation was carried out for on-machine mass cytometry detection.
The analysis results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210375754.X | Apr 2022 | CN | national |
