Analysis of skl mixed culture of lymphocytes. Mixed culture of lymphocytes

Every web developer needs to know SQL in order to write database queries. And, although phpMyAdmin has not been canceled, it is often necessary to get your hands dirty to write low-level SQL.

That is why we have prepared a short tour of the basics of SQL. Let's get started!

1. Create a table

The CREATE TABLE statement is used to create tables. The arguments must be the name of the columns, as well as their data types.

Let's create a simple table named month. It consists of 3 columns:

id– Number of the month in the calendar year (integer).
name– Name of the month (string, maximum 10 characters).
days– Number of days in this month (integer).

Here's what the corresponding SQL query would look like:

CREATE TABLE months (id int, name varchar(10), days int);

Also, when creating tables, it is advisable to add a primary key for one of the columns. This will keep records unique and speed up select queries. Let in our case the name of the month be unique (column name)

CREATE TABLE months (id int, name varchar(10), days int, PRIMARY KEY (name));

date and time

Data type	Description
DATE	Date values
DATETIME	Date and time values with minute precision
TIME	Time values

2. Insert rows

Now let's fill in our table months useful information. Adding records to a table is done through the INSERT statement. There are two ways to write this instruction.

The first way is not to specify the names of the columns where the data will be inserted, but to specify only the values.

This notation is simple, but unsafe, because there is no guarantee that as the project expands and the table is edited, the columns will be in the same order as before. A safer (and at the same time more cumbersome) way of writing an INSERT statement requires specifying both the values and the order of the columns:

Here is the first value in the list VALUES matches the first specified column name, and so on.

3. Extracting data from tables

The SELECT statement is our best friend when we want to get data from the database. It is used very often, so please read this section very carefully.

The simplest use of the SELECT statement is a query that returns all columns and rows from a table (for example, a table named characters):

SELECT * FROM "characters"

The asterisk character (*) means that we want to get data from all columns. Since SQL databases usually consist of more than one table, it is required to specify keyword FROM , followed by a space followed by the name of the table.

Sometimes we don't want to get data from not all columns in a table. To do this, instead of an asterisk (*), we must write the names of the desired columns separated by commas.

SELECT id, name FROM month

Also, in many cases we want the results to be sorted in a specific order. In SQL, we do this with ORDER BY . It can take an optional modifier - ASC (default) to sort in ascending order or DESC to sort in descending order:

SELECT id, name FROM month ORDER BY name DESC

When using ORDER BY, make sure it comes last in the SELECT statement. Otherwise, an error message will be issued.

4. Data filtering

You've learned how to select specific columns from a database using an SQL query, but what if we want to retrieve specific rows as well? The WHERE clause comes to the rescue here, allowing us to filter data based on a condition.

In this query, we select only those months from the table month that are greater than 30 days using the greater than (>) operator.

SELECT id, name FROM month WHERE days > 30

5. Advanced data filtering. AND and OR operators

Previously, we used to filter data using a single criterion. For more complex data filtering, you can use the AND and OR operators and the comparison operators (=,<,>,<=,>=,<>).

Here we have a table containing the four best selling albums of all time. Let's pick the ones that are classified as rock and have less than 50 million copies sold. This can be easily done by placing an AND operator between these two conditions.

SELECT * FROM albums WHERE genre = "rock" AND sales_in_millions<= 50 ORDER BY released

6. In/Between/Like

WHERE also supports several special commands, allowing you to quickly check the most commonly used queries. Here they are:

IN - used to specify a range of conditions, any of which can be met
BETWEEN - Checks if the value is in the specified range
LIKE - searches for certain patterns

For example, if we want to select albums with pop And soul music, we can use IN("value1","value2") .

SELECT * FROM albums WHERE genre IN ("pop","soul");

If we want to get all the albums released between 1975 and 1985, we would write:

SELECT * FROM albums WHERE released BETWEEN 1975 AND 1985;

7. Functions

SQL is chock-full of functions that do all sorts of useful things. Here are some of the most commonly used:

COUNT() - returns the number of rows
SUM() - returns the total sum of a numeric column
AVG() - returns the average value from a set of values
MIN() / MAX() - Gets the minimum / maximum value from a column

To get the most recent year in our table, we must write the following SQL query:

SELECT MAX(released) FROM albums;

8. Subqueries

In the previous paragraph, we learned how to do simple calculations with data. If we want to use the result from these calculations, we cannot do without nested queries. Let's say we want to output artist, album And release year for the oldest album in the table.

We know how to get these specific columns:

SELECT artist, album, released FROM albums;

We also know how to get the earliest year:

SELECT MIN(released) FROM album;

All it takes now is to combine the two queries with a WHERE:

SELECT artist,album,released FROM albums WHERE released = (SELECT MIN(released) FROM albums);

9. Merging tables

In more complex databases, there are multiple tables that are related to each other. For example, below are two tables about video games ( video_games) and video game developers ( game_developers).

Table video_games there is a developer column ( developer_id), but it contains an integer, not the name of the developer. This number is an identifier ( id) of the corresponding developer from the game developers table ( game_developers) linking the two lists logically, allowing us to use the information stored in both of them at the same time.

If we want to create a query that returns everything we need to know about games, we can use an INNER JOIN to link columns from both tables.

SELECT video_games.name, video_games.genre, game_developers.name, game_developers.country FROM video_games INNER JOIN game_developers ON video_games.developer_id = game_developers.id;

This is the simplest and most common JOIN type. There are several other options, but they apply to less frequent cases.

10. Aliases

If you look at the previous example, you will notice that there are two columns called name. This is confusing, so let's set an alias to one of the repeated columns, for example, name from the table game_developers will be called developer.

We can also shorten the query by aliasing the table names: video_games let's call games, game_developers - devs:

SELECT games.name, games.genre, devs.name AS developer, devs.country FROM video_games AS games INNER JOIN game_developers AS devs ON games.developer_id = devs.id;

11. Data update

Often we need to change the data in some rows. In SQL, this is done with the UPDATE statement. The UPDATE statement consists of:

The table containing the replacement value;
Column names and their new values;
The rows selected with WHERE that we want to update. If this is not done, then all rows in the table will change.

Below is the table tv_series with series with their rating. However, a small error crept into the table: although the series Game of Thrones and is described as a comedy, it really isn't. Let's fix this!

tv_series table data UPDATE tv_series SET genre = "drama" WHERE id = 2;

12. Deleting data

Deleting a table row with SQL is a very simple process. All you have to do is select the table and row you want to delete. Let's remove the last row in the table from the previous example tv_series. This is done using the >DELETE statement.

DELETE FROM tv_series WHERE id = 4

Be careful when writing the DELETE statement and make sure the WHERE clause is present, otherwise all table rows will be deleted!

13. Deleting a table

If we want to remove all rows, but leave the table itself, then use the TRUNCATE command:

TRUNCATE TABLE table_name;

In the case when we really want to delete both the data and the table itself, then the DROP command will come in handy:

DROP TABLE table_name;

Be very careful with these commands. They cannot be undone!/p>

This concludes our SQL tutorial! We haven't covered much, but what you already know should be enough to give you some practical skills in your web career.

Co-cultivation of lymphocytes with MHC-II molecules of different haplotype causes their blast transformation and proliferation. Reacting cells belong to T-lymphocytes and are stimulated by foreign MHC-II determinants located on B-lymphocytes, macrophages. The strength of the reaction depends on the degree of difference between the histocompatibility antigens. To assess the reactivity of an individual in relation to the MHC allelic variant, a unidirectional culture of lymphocytes is prepared. To do this, stimulator cells are pre-treated with mitomycin or irradiated, while stimulating cells lose their ability to divide, but remain viable.

Reactivity in a mixed culture of lymphocytes is one of the criteria in the evaluation cellular immunity. SKL allows to carry out HLA typing, to select a pair of donor-recipient for tissue and organ transplantation, to make a correlation between HLA antigens and certain diseases.

To set up the reaction, a suspension of lymphocytes isolated from the blood of patients (donor and recipient) is used. The washed responding cells are suspended in a small amount of medium 199 (0.5 ml) and, after counting the number of cells, adjusted to 0.6 * 10 6 cells/ml. The stimulating cell suspension is prepared similarly with the addition of a solution of mitomycin C (500 μg of mitomycin C in 1 ml of buffered saline) at a rate of 0.1 ml per 1 ml of suspension. The cell mixture is incubated for 40 minutes at 37°C in a water bath with stirring, then cold medium 199 is added to the suspension and the cells are washed three times by centrifugation. The precipitate is suspended in culture medium cultivation, bringing the number of cells to 0.6 * 10 6 cells/ml.

0.1 ml of the cell suspension in Hank's solution is added to the wells of a sterile round-bottomed immunological plate, and 0.1 ml of the cell suspension carrying MHC allelic variants is added. The same amount of Hank's solution was added to the control.

Accounting for the results is carried out after incubating the cells at 37 ° C for 3-5 days, depending on the formulation of the reaction using a morphological or isotope method (see RBTL).

When transplanting bone marrow the choice of a histocompatible donor is based on the results of HLA typing of the donor and recipient, as well as when determining the compatibility of selected HLA-identical donor-recipient pairs in the SCL, which is the final stage of compatibility assessment. For this, a bidirectional SCL reaction is used using a microcultural method in a 96-well plate with an assessment of the magnitude of proliferation by the incorporation of radioactive H 3 -thymidine into the DNA of proliferating lymphocytes.

Much attention is currently being paid to the study of the role immune mechanisms in the reproductive process, since their violation leads to the development of infertility and early termination of pregnancy. Studies of immunological parameters and their relationship with reproductive processes led to the conclusion that restructuring is necessary immune system in preparing the mother's body for pregnancy, starting with ovulatory period, at the time of embryo implantation and at early period pregnancy.

One of the most complex and difficult to diagnose causes of infertility and recurrent spontaneous miscarriages is immune dysfunction.

With the aim of timely detection immunological infertility in the laboratory of immunology of reproduction is being examined couples using following methods diagnostics:

study of the level of proliferative response of a woman's lymphocytes to partner antigens (in a mixed culture of lymphocytes)
determination of the activity of blocking factors in the blood serum of a woman.
assessment of the quantitative content of subpopulations of natural cytotoxic cells in peripheral blood women.
expanded immunogram
study of the content of regulatory cells with suppressor activity in peripheral blood.
when identifying clinical and laboratory signs immunological infertility, patients are recommended to undergo medical procedure- alloimmunization with husband's lymphocytes, which is carried out in accordance with an individual schedule.

Consultation needed:

if in the absence of gynecological and endocrine diseases with regular sex life during the year there are no pregnancies.
in case of repeated (more than 1 time) spontaneous abortions (miscarriage).
at unsuccessful IVF in history.
with the threat of termination of a real pregnancy.

If violations are detected, a procedure is prescribed for the purpose of immunocorrection of violations. alloimmunization with partner lymphocytes (AIL). This method treatments are approved Federal Service on supervision in the field of healthcare (license FS No. 2009/179 dated 02.07.2009)

The procedure is carried out only if the partner has negative results testing for HIV infection, syphilis and viral hepatitis(B, C). Complications in AIL were not identified. The AIL method has a pronounced immunocorrective effect, increases the effectiveness of infertility treatment, as in natural cycle, as well as in vitro fertilization(ECO). It is also prescribed for unsuccessful attempts at in vitro fertilization.

AIL is carried out 1 time in 28-30 days (in accordance with menstrual cycle women) in the 1st phase of the cycle. The first course includes 3 AIL procedures. After the 2nd procedure, the couple retakes the control analysis. In the presence of positive dynamics, the third AIL procedure is the last one. In the absence of dynamics, the dose of cells for immunization is increased and the 2nd course is carried out, which includes 2 procedures. After a control analysis, in some cases, it is necessary to prescribe a 3rd course of immunization with an increase in the dose of cells. After reaching the standard values, the positive effect lasts within 6-8 months.

For the period from 2003 to 2013. in the laboratory of cellular immunotherapy, about 3000 procedures of alloimmunization with partners' lymphocytes were carried out. For this procedure, only lymphocytes are isolated from the partner's blood, which are always present in the semen. In other countries*, the alloimmunization procedure has also been carried out for 20 years, which helps a woman bear and give birth to a healthy child.

Type of service	price, rub.
Lymphocyte transformation test	1650
Lymphocyte transformation test (Detection of sesibilization on 2v-va)	2 900
Lymphocyte transformation test (Detection of sesibilization on 3v-va)	3 900
Lymphocyte transformation test (immunological examination for infertility)	3 200
Expanded immunogram with activation markers	4 625
Comprehensive study of the immune status	4 125
Vaccination (alloimmunization with partner's lymphocytes 1 dose)	1 900
Vaccination (alloimmunization with partner's lymphocytes 2 dose)	2 750
Vaccination (alloimmunization with partner's lymphocytes 3 dose)	3 400
Investigation of CD16+/CD56+ lymphocytes (immunological examination for infertility only NK cells)	1 250
Study of macrophage activity (conditioned environments of macrophages)	9 400
Study of macrophage activity (conditional media)	1 500
Cryopreservation of mononuclear cells for alloimmunization	650

2.2. Methods of cell interactions

Almost all methods of cell interactions used in clinical immunogenetics are based on the phenomenon of blast formation in a mixed culture of lymphocytes, discovered by Canadian researcher Barbara Bain. The mixed lymphocyte culture (MLC) reaction allowed for a finely differentiated study of the HLA complex and, in particular, one of its subunits, the HLA-D locus. A number of other methods of cell interactions - cell-mediated lympholysis (Cell-mediated Lympholysis - CML), the test of priming lymphocytes (Primed Lymphocyte Typing) - is based on the MLC method or contains it as an integral part.

2.2.1. Mixed culture of lymphocytes (MCL)

The principle of the method is shown in fig. 10, which shows that two populations of genetically different lymphocytes interact with each other in in vitro culture. One of the populations is treated with mitomycin C or irradiated, as a result of which it loses the ability to blast formation and thymidine incorporation (3 HT), however, without losing antigenic properties, it retains the ability to stimulate (stimulants; S-population).

Cells that respond to stimulation (responders, R-population) transform into blasts after a few days and include thymidine added to the culture. The intensity of the reaction is measured using a radiation label by the incorporation of 3 H-thymidine.

Several variants of the reaction of a mixed culture of lymphocytes are known, of which two are proposed here: a traditional one and a microvariant implemented in Cook plates (Fig. 11).

Rice. 11. Equipment for microvariants MLC and CML. 1 - tablet round bottom e 96 holes; 2-automatic pipette ("Pipetman") with program for different volumes; 3 - automatic pipette ("Sigma") for a certain volume; 4 - disposable pipette tip; 5 - laminar box

Traditional version of MLC (modified by F. S. Baranova):

Lymphocytes are isolated on a ficoll-urographin gradient as described above. The first wash is carried out in PBS (NaCl - 8.0 g, Na 2 HP0 4 - 1.15 g, KH 2 PO 4 - 0.2 g, KC1 - 0.2 g per 1 liter of H 2 O); the next two are produced in medium 199 with 5% AB serum (pool of 15 donors);

Responding cells are resuspended in 199 medium with 10% AB serum and the cell concentration is adjusted to 0.6 x 10 6 in 1 ml;

Stimulating cells isolated in the same way at a concentration of 5 - 10 6 per 1 ml are treated with mitomycin C (60 γ/ml) from Serva and incubated for 40 minutes at 37°C in a water bath. After incubation, the cells are washed 3 times with medium 199 with 5% serum AB, resuspended in medium 199 with 10% serum AB, bringing the concentration to 0.6 x 10 6 in 1 ml;

0.5 ml of responding cells and 0.5 ml of stimulating cells are mixed in a centrifuge tube, 0.04 ml of 10 ml of Hepes solution (Serva) is added and incubated for 144 hours; experience is put in three parallels (triplet);

24 hours before the end of the incubation, 3 H-thymidine 1μCi (3.7x10 4 BC) is added to the tubes, per sample specific activity 5mSi/mmol (18.5x10 7 Bq/mmol) and continue incubation;

At the end of the incubation, the cells are transferred to millipore filters (0.6 - 0.9 microns), washed saline(37°C) and chilled 5% trichloroacetic acid solution (4°C). The filters are dried and placed in vials with 5 ml of scintillation fluid (5 g of PPO and 0.5 g of POPOP per 1 liter of toluene)*. 3 H-thymidine incorporation activity is measured on a β-counter; the result is expressed in absolute inclusions of a radioactive label in 1 min or in the stimulation index (SI) according to the formula:

* (PPO - 2,5-phenol-oxazole; POPOP - (1,4-di-2-15-phenol oxazolitebenzene).)

Average value of CPM in three prototypes

SI = Mean CPM of three controls/Controls are spontaneous cultures of responding cells.

MLC micro variant in Cook tablets. At the 8th Workshop, requirements were developed that standardize the MLC micro-variant, in accordance with which it is presented below:

Lymphocytes are isolated in an isopac-ficoll gradient and resuspended in RPMJ-1640* medium, bringing the concentration to 5x10 5 in 1 ml;

* (Medium 199 mixed with group AB human serum (5% serum taken from several individuals) can be used.)

Stimulator cells are treated with mitomycin C or training;

5x10 4 reponders and the same number of stimulants are placed using a Pipetman type micropipette into each well of a Cook round-bottom microplate;

The plates are incubated in a thermostat with automatic supply of 5% CO 2 for 120 h; then lμmCi 3 H-thymidine is added to each well at a thymidine specific activity of 6 Ci/mmol; all manipulations are carried out with a Pipetman pipette;

16 hours after adding thymidine, the cultures from each well are transferred to millipore filters with an automatic pipette and washed with saline and then with 5% trichloroacetic acid; it is convenient to use special "harvesters" to transfer the culture to millipore filters;

Thymidine incorporation activity is determined as described above.

2.2.2. Cell mediated lympholysis (CML)

CML has been used in immunogenetic studies relatively recently (since 1972), but in Lately becomes more and more popular technique, allowing you to study in detail the role of the efferent link of transplant immunity and gaining recognition as informative method immunological monitoring. The principle of the method is shown in fig. 12. The method is based on the technique proposed by J. Lightbody (1971), which is summarized as follows.

1. CML begins with a conventional HLC test in which responder cells recognize "stimulators", sensitize responder cells, and form sensitized killers.

2. The second phase, in which sensitized killers are mixed with 51 Cr-labeled target cells, consists in the destruction of the latter by effector killer cells; target cells may have the same genotype as the "stimulators" in the first phase of MLC, or they may be cells of the "third" partner, endowed with antigenic determinants in common with the "stimulators" or without them.

3. The amount of radioactive chromium released from the destroyed target cells and released into the culture medium serves as a measure of the intensity of the killer effect.

Rice. 12. The principle of cell-mediated lympholysis (CML). I row - sensitization of responding cells, the formation of killers; II row - the interaction of killers with the target; III row - destruction of the target cell; exit 51 Cr culture medium

Three variants of CML are described here: the traditional version modified by L.P. Alekseev (1979), the micro-variant in Cook plates, direct CML (Direct-CML -D-CML.

Traditional option[Alekseev L.P., 1979].

The method is divided into several stages.

Getting hitmen:

Peripheral lymphocytes of both populations (R-cells and S-cells) are isolated in the usual way, washed in a density gradient three times in medium 199 with 5% AB serum (10 min, 150 g);

1x10 6 R cells (0.8 ml) are mixed in centrifuge tubes with 2x10 6 S cells (0.2 ml) treated with mitomycin C and incubated for 144 h at 37°C;

The cells are centrifuged at 150 g for 10 min, and the pellet is resuspended in medium 199 with the addition of 20% calf serum (medium 199 + i.e.), bringing the concentration to 1x10 6 in 1 ml;

One of the samples is left to study the level of MLC, the rest are used as ready-made killers.

Receiving targets:

Conventionally isolated lymphocytes are resuspended in 199 medium with 20% AB serum and incubated with phytohemagglutinin (0.003 mg/ml Wellcome) for 72 hours at 37°C; cell concentration 1x10 6 in 1 ml;

Cells are centrifuged for 10 min at 150 g, the pellet is resuspended in medium 199 with 5% AB serum, and the cell concentration is adjusted to 2x10 4 in 1 ml;

51 Cr 100 μCi/ml (3.7x10 6 Bq/ml) is added to the suspension and incubated for 40 min at 37°C;

Cells are washed three times in 199 medium supplemented with 5% AB serum (150 g, 10 min) and the pellet is resuspended in 199 medium supplemented with 5% AB serum. bringing the concentration to 2x10 4 in 1 ml.

Statement of reactions:

5x10 5 killers (0.5 ml) are mixed with 1x10 4 targets (0.5 ml), incubated for 4 h (37°C) and centrifuged at 150 g for 10 min;

The supernatant is sucked off and the impulse caused by 51 Cr is counted on a?-counter; counting is carried out in 0.5 ml of supernatant;

The level of cytolysis is calculated by the following formula:

ekeper. output 51 Cr - spontaneous output 51 Cr/max, output 51 Cr - spontaneous output 41 Cr × 100.

Spontaneous release of chromium is measured on targets incubated for 4 hours (37°C) without killer cells; maximum yield of chromium - on targets completely destroyed by freezing-thawing; All tests are carried out in triplets.

Microvariant of CML in tablets [according to Mawas S., 1976]:

The killer production phase is carried out as MLC in round bottom plates, but R- and S-cells are mixed in equal amounts at 2x10 5 PA each well and incubated at 37°C;

After 5 days, the cells are collected with a Pasteur pipette in a test tube, the number of living cells is counted with trypan blue, and the concentration is adjusted to 10x10 6 in 1 ml;

Target cells are cultured with PHA for 3 days;

On the day of the reaction, the target cells are washed (300 g) and resuspended in the culture medium, distributing 1 ml into test tubes and bringing to 1x10 6 in 1 ml;

200 μCi 51 Cr (7.4x10 6 BK) are added to each tube with target cells and incubated for 1 h at 37°C; wash the cells 3 times; the sediment is resuspended and adjusted to a concentration of 1x10 in 1 ml (live);

The test is implemented in round-bottom microplates; for this, 0.7 - 1x10 6 killer cells (0.1 ml) and 1x10 4 target cells (0.1 ml) are distributed into each well; the total volume of the suspension in each well is 0.2 ml, 0.05 ml of the medium is added to each well and incubated (37°C) for 4 h;

The plates are centrifuged at 800 g in a Beckman centrifuge for 10 minutes; the supernatant from each well is transferred into test tubes and counted on a γ-counter;

The killer production phase (MLC) is carried out on RPMJ-1640 medium supplemented with 20% human plasma; for the killing phase, MEM medium is used with the addition of 20% human plasma, preheated.

Direct CML (D-CML). Direct CML is a technique developed specifically for clinical purposes that has found use in immunological monitoring. The scheme of this reaction is shown in fig. 13.

The main difference from traditional CML is that the stage of killer formation (sensitization phase) does not occur in vitro, but in vivo, i.e., in the human body sensitized by transplantation of an allogeneic organ or tissue. Due to the impact of allogeneic tissue, the recipient's lymphocytes are sensitized to the donor's tissue antigens and do not need special in vitro treatment, the test duration is significantly reduced in time, since only the targets should be prepared first.

The targets are lymphocytes obtained from the peripheral blood or, more commonly, from the spleen of a donor (see 2.1.2) and frozen by any of the methods described above (see 2.1.3).

2.2.3. Antibody-dependent-cell mediated Cytotoxicity (ADCC)

This type of cellular response is similar in mechanism of action to the CML described above. The principle of the reaction is shown in fig. 14. The components of the reaction are target cells (donor cells or allogeneic lymphocytes), serum (allogeneic or recipient), presumably containing antibodies directed to target cell determinants, and effector cells (recipient lymphocytes or allogeneic lymphocytes).

Effector cells in this type of interaction are the so-called K-cells located in the peripheral blood. Unlike killer cells in CML, they carry out a cytotoxic effect without prior sensitization, however, the manifestation of the cytotoxic effect of K cells is possible only through antibodies directed to the determinants of target cells. Specific lysis is measured by the release of 51 Cr if the test serum contains antibodies to the target determinants.

Target determinants in ADCC can be specificities of almost all HLA loci, including HLA-D, HLA-DR, as well as determinants outside the HLA system.

Most widespread this complex reaction was obtained in immunological monitoring to detect B-cell antibodies. In this regard, several modifications have been proposed, involving enrichment of the target cell suspension with B-lymphocytes, adsorption of sera on platelets, etc.

In addition, a number of other features of the response in this system are taken into account. The test serum must be decomplemented, otherwise the reaction may proceed as an antibody-mediated complement-dependent cytotoxicity. It is also assumed that effector cells can cause certain destruction of targets by the CML type.

Ultimately, the entire set of samples in the test of antibody-dependent cell-mediated lymphocytotoxicity is as follows:

Targets + effectors + tested serum (experimental sample);

Targets (control sample, i.e. spontaneous release of 51 Cr);

Targets + effectors (control test for effector activity);

Targets + test serum (serum control).

Lymphocytes are isolated in the usual manner and resuspended in RPMI-1640 + 10% fetal bovine serum; treating the suspension with carbonyl iron to remove monocytes; add ammonium chloride or H 2 O to lyse the remaining erythrocytes;

51 Cr is added to the target cell suspension in the form of Na 2 CrO 4 solution 100 - 200µCi ml (3.7x10 6 - 7.4x106 BK / ml) and incubated for 40 - 60 minutes;

Cells are washed three times in RPMI + 0.1% HSA, resuspended in the same medium and adjusted to 2x10 8 in 1 ml; 50 µl of the labeled cell suspension is placed in a test tube and the isotopic activity is then counted on a γ-counter to detect complete isotopic "uptake"; the rest is dropped into the wells of the tablet at 50 µl of suspension (1x10 5 cells per well);

Suspension of cells-effectors is brought to 2x10 7 cells in 1 ml and in. put 50 µl of suspension (or 100 µl) into each well, effector to target ratio 10:1 (or 20:1);

The incubation lasts 4 hours in a humid atmosphere incubator with 5% CO 2 (the duration of the incubation can be extended up to 8 hours);

Prepare in test tubes several dilutions of the test serum (1:25; 1:100) and add to the wells up to 50 µl of each dilution: and undiluted serum; The final setting of the test looks as shown in Table. 23.

Table 23

Setting ADCC. All sample options, µl

* (Instead of the test serum, a pool of AV sera is instilled.)

** (Instead of the test serum, a monospecific HLA serum directed against targets (not effectors) is instilled.)

The panels are incubated for 4 hours in a humid atmosphere with 4% CO 2 ; the duration of incubation can be extended up to 8 hours;

After incubation, half of the volume from each well (100 μl) is carefully aspirated with an automatic pipette and placed in tubes for counting 51 Cr impulses; activity is counted on a γ-counter;

The calculations are as follows:

% release of 51 Cr = 2 × A op - background activity/B - background activity × 100;

% cytotoxicity = A op - A sp /100 - A sp × 100, where A op - % release of 51 Cr in experimental samples; And cn -% spontaneous release; B - complete assimilation of the isotope.

How positive result 5% or higher cytotoxicity after 4 h of incubation is considered.