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Isolation, Propagation, and HIV-1 Infection of Monocyte-Derived Macrophages and Recovery of Virus From Brain and Cerebrospinal Fluid — Микроскопическая техника в биологии и медицине

Isolation, Propagation, and HIV-1 Infection of Monocyte-Derived Macrophages and Recovery of Virus From Brain and Cerebrospinal Fluid

Santhi Gorantla, Myhanh Che, and Howard E. Gendelman

Summary

Mononuclear phagocytes (MP: monocytes, dendritic cells, and tissue macrophages) are host cells for the human immunodeficiency viruses types 1 and 2. MPs are both the first lines of defense and vehicles for viral dissemination in the infected human host. Viral infection of MP can affect the disease directly during interstitial pneumonitis and HIV encephalitis. Both revolve around MP secretions of immune regulatory and neurotoxic factors. Clearly, laboratory models that mimic disease need to include primary human MP infected with viral isolates obtained from diseased tissues. Over the past two decades our laboratory has developed state-of-the-art methods for isolation and propagation of monocytes from peripheral blood. This technology directly supports work at the University of Nebraska Medical Center as well as research performed throughout the United States, including the laboratories of Drs. Mario Stevenson, William Tyor, David Volsky, Loyda Melendez, and Mary-Jane Potash, among others. The importance of these cells as targets for virus and reservoirs of persistent infection are discussed.

Key Words: Monocytes; macrophages; elutriation; HIV-1 infection; mycoplasma; endotoxin contamination.

1.  Introduction

Cells of mononuclear phagocyte lineage (MP: monocytes, dendritic cells, and tissue macrophages) are primary cellular targets for HIV infection and viral dissemination (1-4). They also serve as reservoirs of virus throughout body compartments including, most notably, peripheral blood, lung, and the central nervous system (CNS) (5-7). Macrophage-tropic HIV-1 can be propagated directly onto monocyte-derived macrophages (MDM) from infected body

fluids and tissues including plasma, blood, lung, brain, and cerebrospinal fluids (8,9). Monocytes can be maintained in culture for more than 3 mo and thus provide susceptible targets for the propagation of virus from patient samples. Permissiveness of macrophages to productive HIV infection depends on the differentiation state of the cells (10). Viability and differentiation of monocytes isolated from peripheral blood are maximized when cultivated with recombinant human macrophage colony stimulating factor type 1 (referred to as MCSF). Such control conditions also permit increased susceptibility to HIV-1 infection (9).

Viral recovery from infected brain samples or cerebrospinal fluid directly onto MDM is appropriate as MP disseminate virus to the CNS and are productively infected during the disease. Moreover, viral production is often continuous in MP. Macrophage indicator systems represent adherent cell targets where virus-induced cytopathicity is readily observed. Isolation of monocytes by countercurrent centrifugal elutriation allows the isolation of a large number of relatively pure cell populations (11). Alternatively, isolation of cells by adherence or gradient systems diminishes cell purity and may change the cell phenotype (12). In this chapter, we describe the isolation of monocytes as well as quality control measures for achieving optimal cell culture conditions free of microbial contamination for the purpose of studies of HIV-host cell interactions as they may occur during disease.

2.            Materials

2.1.        Isolation and Propagation of Human Monocytes

1. Leukocyte-enriched whole blood (leukopak), from HIV-1,-2 and hepatitis B seronegative donors, is obtained from a blood bank and processed within 6 h of collection.
2. Lymphocyte separation medium (LSM).
3. Phosphate-buffered saline (PBS), filter-sterilize through 0.2-pm filter unit or autoclave for sterilization.
4.  Elutriation, J6-MI centrifuge with a JE-5.0 elutriation system (Beckman, Fullerton, CA).
5.  Cell counter and size analyzer (Beckman Coulter, Fullerton, CA).
6.  Isoton diluent (Beckman Coulter).
7. Percoll gradient (1.087 g/mL): 12.5 mL of Percoll, 5.5 mL of sterile distilled water, 2 mL of 10X PBS.
8.  Dulbecco’s minimal essential medium (DMEM) without L-glutamine
9.  Human AB serum (HS), heat inactivated at 56°C for 30 min.
10. L-glutamine.
11. Gentamicin.
12.  Ciprofloxacin.
13.  Recombinant human MCSF.
14.  Monocyte medium without MCSF: DMEM, 10% HS, 2 mML-glutamine, 50 pgl mL gentamicin, 10 pg ciprofloxacin. Filter-sterilize using 0.2 pm filter unit. Store at 4°C. Discard after 2 wk.
15. Monocyte medium with MCSF: DMEM, 10% HS, 2 mM L-glutamine, 50 pglmL gentamicin, 10 pg ciprofloxacin, 1000 UlmL rhMCSF-1. Filter-sterilize using 0.2-pm filter unit. 16. Store at 4°C. Discard after 2 wk.
16.  Trypan blue solution, 0.4 % in saline or PBS.
Aliquot reagents 8, 9, 11, and 12 for use in 500-mL bottles of DMEM. Reagents are stored at -20 °C and freeze-thaw cycles are minimized.

2.2.        Propagation of HIV-1 From Brain Tissue

1.            Kevlar gloves or cut-resistant gloves.
2.            Scalpels and forceps, sterile.
3.            Monocyte medium with and without MCSF (as described in Subheading 2.1.).

2.3.        Reverse Transcriptase (RT) Assay

1. Solution A (disruption buffer): 100 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM dithiothreitol (DTT), 0.1% NP40.
2. Solution B (RT reaction mix): 50 mM Tris-HCl, pH 7.9, 150 mM KCl, 5 mM DTT, 15 mM MgCl2, 0.05% NP40, 10 pglmL Poly A, 0.25 UlmL oligo dt pd(T)12-18, 4 pCilmL 3H-thymidine (add 3H-thymidine just before use; 3H-thymidine is radioactive and must be handled carefully according to radiation safety procedures). Solution A and solution B can be stored as aliquots at -20°C for up to 6 mo.
3.            10% trichloroacetic acid (TCA).
4.            Standard RT enzyme.
5.            Cell harvester.
6.            Liquid scintillation counter.

 

2.4.  Screening for Mycoplasma and Endotoxin Contamination

1. Pyrotell Limulus Amebocyte Lysate (LAL) (Associates of Cape Cod, Inc., East Falmouth, MA).
2.  Control standard endotoxin (CSE) (Associates of Cape Cod).
3.  Pyrotubes (Associates of Cape Cod).
4.  LAL reagent water (Associates of Cape Cod).
5. Triton X-100.
6.  Tributylphosphate.
7. Mycoalert (Cambrex Bio Science Rockland, Rockland, ME).
8. Luminometer.

3.            Methods

3.1.        Isolation of Monocytes From Peripheral Blood

Countercurrent centrifugal isolation is performed on leukocyte-enriched blood samples of approx 100 mL. The yield of monocytes by this method is 1.5  to 2 billion cells. An alternative method to isolate monocytes from smaller volumes (<50 mL) of blood is by Percoll gradient separation or by adherence. In this section we describe all three methods for monocyte isolation.

3.1.1.     Donor Screening

Donors are screened for HIV-1 and 2 and hepatitis B and C. Leukopaks are obtained only from seronegative individuals for monocyte isolation. The donors are screened for ABO Rh and antibodies against hepatitis B surface (HBS) or core antigen (HCV), hepatitis C virus (HCV), HIV, human T lymphotropic virus (HTLV), cytomegalovirus (CMV), and syphilis per the institutional regulations.

Donors are chosen without heart, lung, and liver disease. Those with anemia regardless of cause and pregnancy are excluded. Donors undergo a physical examination by a physician before being qualified for leukophoresis. On the day the procedure is performed, the donor’s hematocrit and platelet count are checked and must be >38% and 150 x 1010/mL, respectively.

3.1.2.     Countercurrent Centrifugal Elutriation

The procedure is carried out in a biological safety cabinet (BSC) under sterile conditions (see Note 1); biosafety level 2 (BSL-2) practices are required for handling human specimens, given the risks associated with their use. BSL-2 and BSL-3 practices are followed per National Institutes of Health guidelines; these include wearing laboratory coats and gloves and decontaminating liquid waste with 50% bleach. All materials are autoclaved before being discarded.

 

3.1.2.1. Preparation of Elutriation Setup

1.   Prepare the elutriation setup before starting to process the blood sample.

2.  Assemble the chambers and the rotor per the instrument instruction manual.

3.   Connect the input tubing and the output tubing to the rotor chamber.

4.  The input tubing is connected through a peristalitic pump that controls the flow rate. To the other end of the input tube connect a three-way stopcock, which is used to control the input of either PBS or cells pumped from two separate reservoirs (the manual provides detailed instructions for the setup). The output tubing goes directly into a waste container while collecting washes, or a sterile bottle while collecting the cells (see Note 2).

5. Sterilize the chamber and the tubing by pumping 95% ethanol (approx 200 mL) through the system (see Note 3).

6.  Fill PBS in both reservoirs and flush the system with PBS to remove air bubbles. Use at least 400 mL of PBS.

7.  Flush PBS through the system by increasing the pump setting to maximum. This is to ensure that the setup can handle the pressure. Check the system for any air bubbles and prevent them by flushing the system again with more PBS.

8.  Now proceed to processing the leukopak.

 

Fig. 1. Lymphocyte separation medium (LSM) gradient. Separation of whole blood using LSM and showing a buffy coat at plasma and media interface.

3.1.2.2. Isolation of Monocytes

1.  Transfer the blood sample from the leukopak into a 250-mL centrifuge tube. Use sterile scissors to cut the leukopak. Dilute the blood sample by adding enough PBS to make up to a total of 250 mL. Pipet up and down to mix gently.
2.  Aliquot 10 mL of LSM into each of the 10 50-mL centrifuge tubes.
3. Layer 25 mL of diluted blood sample carefully onto the LSM without disturbing the interface. To maintain the interface, the tube is held at a 45° angle.
4.   Centrifuge at 600g at room temperature for 20 min without brake.
5.  After centrifugation, transfer the tubes from the centrifuge to the BSC without disturbing the gradient. Red blood cells settle at the bottom and the peripheral blood mononuclear cells (PBMC) form a buffy coat layer at the LSM and plasma interface. This is shown in Fig. 1. Aspirate the top layer containing diluted plasma and platelets directly above the buffy coat.
6. Collect the buffy coat containing PBMC from each of the gradient tubes and transfer the recovered cells to a fresh 250-mL centrifuge tube.
7.  Make the volume up to 250-mL by adding PBS. Mix well by gently pipetting up and down.
8. Centrifuge for 10 min at 400g at room temperature.
9. Decant supernatant and resuspend the cell pellet in 250 mL of PBS and repeat step 8. Finally, suspend the cells in 25 mL of PBS.
10. Replace one of the reservoirs in the elutriation setup with the tube containing the cell suspension.
11. With PBS flow open (controlled with the stopcock), turn on the pump at a flow rate of 40 mL/min. Let PBS flow through the system.
12. Turn on the centrifuge and when the rotor reaches 1960 rpm (see Note 4), stop PBS flow and load the cells by turning the stopcock. After all the cells are loaded, turn the stopcock back to allow PBS flow. Wait for 10-15 min to allow the cells to separate by size within the rotor chamber.
13. Turn the pump to a flow rate of 45 mL/min and wait for 2 min to remove the remaining red blood cells.
14. Turn the pump to 50 mL/min and collect peripheral blood lymphocytes (PBL).
15. Collect aliquots of cell suspension (10 pL) at each speed setting of the pump. Dilute the cell aliquot with 10 mL of isoton diluent. Count and analyze the cells using an electronic counting device (Beckman Coulter cell counter and analyzer), which also gives a profile indicating the cell size. Calibrate the cell analyzer with latex beads of known size before analyzing the cells. Lymphocyte profile will be that of approx 6-8 pm-diameter cells. Use the cell counter according to the manufacturer’s instructions (see Note 5).
16. Turn the pump up to 55 mL/min to collect more PBL. Count the cells; if it shows >500 cells/mL increase the pump flow by 5 mL/min at a time until all the PBL are eluted.
17. When a profile of monocytes starts showing (refer to the cell analyzer manual; the profile of cells should be same as that obtained for cells of 8-10 mm diameter), stop the centrifuge, set the pump to 200 mL/min, and collect all the monocytes.
18. Centrifuge tubes with PBL or monocytes at 400g for 10 min at room temperature. Decant the supernatant and resuspend the cells in 25 mL of PBS.
19. Use a small aliquot (10 mL) to count the cells and calculate the total number of cells.
20. To check the purity of PBL and monocyte populations, make a cell smear on a glass slide using a small aliquot from each cell suspension and stain with hematoxylin and eosin (H&E). Observe under the light microscope. Figure 2 shows H&E-stained monocytes and PBL separated by this procedure. Monocytes have distinct kidney-shaped nuclei and can be easily identified.

3.1.3.     Percoll Gradients

1. Dilute blood sample 1:1 with PBS.
2. In 15-mL conical tubes take 4 mL of Percoll, adjusted to 1.087 g/mL density, and layer 8 mL of diluted blood carefully over the Percoll gradient without disturbing the interface.
3. Centrifuge the tubes at 600g at room temperature for 30 min without brake.
4. Red blood cells will settle in the bottom and PBMC will form a buffy coat layer at the interface of the Percoll (bottom layer) and the plasma (top layer). Aspirate the plasma carefully up to the buffy coat layer without disturbing it. Transfer the buffy coat into a fresh 15-mL conical tube and dilute with 10 mL PBS.

Fig. 2. Lymphocytes and monocytes isolated by elutriation. Cell fractions of peripheral blood lymphocytes (A) and monocytes (B) isolated by countercurrent centrifugal elutriation are stained with H&E, original magnification x40.

5.            Spin the tubes at 400g for 5 min and decant the supernatant.

6.            Suspend the cells in 10 mL of PBS and repeat step 5.

7.            Finally, suspend the cells in 5 mL of monocyte PBS and count the cells by using trypan blue and a hemocytometer.

3.1.4.     Adherence

1.            Dilute the blood sample 1:1 with PBS.

2.            In 15-mL conical tubes take 4 mL of LSM and layer 8 mL of diluted blood without disturbing the interface.

3.            Centrifuge the tubes at 600g for 20 min at room temperature without brake.

4.            Collect the buffy coat as described in Subheading З.1.2.2., step 5.

5.            Dilute with 10-15 mL PBS and centrifuge the tubes at 400g for 10 min.

6.            Resuspend the pellet in 10 mL PBS by pipetting up and down gently and repeat step 5.

7.            Suspend the pellet in 10 mL monocyte medium without MCSF. Take a 10-pL aliquot to count the cells using trypan blue and a hemocytometer.

8.            Adjust the cell suspension to 2-3 x 106 cells/mL and add an appropriate volume of cell suspension to the culture dish. Take 20 mL in a 75 cm2 flask, 5 mL/well in a 6-well plate, 2 mL/well in a 24-well plate, 1 mL/well in a 12-well plate, or 200 pL/well in a 96-well plate. Choose the size of the culture dish according to the cell number and also how it will be used in further experiments (see Note 6).

9. Incubate the culture at 37°C with 5% CO2 and 95% humidity for 2-3 h.

10.          After 2-3 h cells will adhere to the plastic. Aspirate the supernatant containing nonadherent cells without disturbing the adherent cells. Wash the cells at least 4 times by adding fresh monocyte medium without MCSF without disturbing the adhered cells, swirl the flask, and aspirate the medium to remove as many lymphocytes as possible.

11.          Replace with the same amount of fresh monocyte medium with MCSF. Add medium to the sides of the culture dish to prevent cells from coming off of the surface. Incubate the culture under the same conditions (step 9).

12.          On d 3 and d 5, feed the cells with fresh monocyte medium containing MCSF by doing a half-exchange as described in Subheading 3.2., step 5.

13.          On d 7, cells can be used for HIV-1 infection.

3.2.        Monocyte Propagation

1.            Suspend monocytes in monocyte medium with MCSF at 1 x 106 cells/mL. Gently mix the cells by pipetting to obtain uniform suspension. Plate the cells immediately after elutriation.

2.            Plate an appropriate volume of cells for type of plate or culture dish (see Table 1).

3. Incubate at 37°C with 5% CO2 and 95% humidity.

4.            On d 3, add additional monocyte medium with MCSF (as shown in Table 1) and incubate under the same conditions. By this time cells adhere to the bottom of the culture dish, forming a monolayer.

5.            Half-exchange with fresh medium every 2 d as follows: remove half of the total volume of medium from the culture, without disturbing the cells (see Note 7), and replace with an equal volume of monocyte medium with MCSF.

6.            From d 7, use monocyte medium without MCSF (see Note 8).

Table 1

Cell Number and Volume of Medium

To Be Used According to Size of Culture Dish

Culture dish Cell no. (x106) Media added d 3 (mL)
75-cm2 flask 30/flask in 20 mL 15
6-well plate 3/well in 3 mL 2
24-well plate 0.75/well in 0.75 mL 0.75
48-well plate 0.25/well in 0.25 mL 0.25
96-well plate 0.1/well in 0.1 mL 0.1

3.3.        HIV Infection of MDM

All procedures handling infected specimens should be carried out in at least BSL-2 containment facilities. BSL-3 is recommended for handling high-titer virus preparations.

3.3.1.     Laboratory HIV-1 Isolates

Viral production stays in an exponential state depending on the size of the culture. In a 6-well plate it will be from approx d 7 to d 21. In a 75-cm2 flask there is a higher cell number; therefore, the viral production will be in an exponential state for up to 1 mo. The size of the culture to be used for infection depends on the amount of viral stock needed.

1.            Culture monocytes in an appropriate dish as described in Subheading 3.2.

2.            On d 7, remove all medium from the culture and infect with HIV stock diluted in monocyte medium without MCSF at 0.01 multiplicity of infection (MOI). Refer to Table 2 for the volume of medium containing the virus to be used for each type of culture dish (see Notes 9 and 10).

3.            Incubate at 37°C with 5% CO2 and 95% humidity for 4 h.

4.            Add monocyte medium without MCSF (see Table 2 for the volume), without removing the virus, and incubate overnight under the same conditions.

5.            The following morning, remove all the infection medium from the culture and add the same amount of fresh monocyte medium without MCSF.

6.            On d 10, collect culture supernatant during half-exchange of the media (as described in step 5 of Subheading 3.2.) instead of discarding. Reserve 30-50 p,L for RT assay and store the rest at 4°C (see Note 11).

7.            Repeat step 6 on d 12, 14, 16, 18, 20, 22, and 25 (see Note 12).

8.            Obtain the RT results (perform RT assay as described in Subheading 3.4.) and pool the supernatants with peak RT values.

9. Centrifuge at 300g for 15 min at 4°C, remove the supernatant, and aliquot into cryovials for storage either at -80°C or in a liquid nitrogen tank (see Note 13).

HIV-infected MDM form multinucleated giant cells (fusion of infected and uninfected cells, Fig. 3B). HIV-infected MDM express HIV-p24 (Fig. 3D).

Fig. 3. Cytopathicity of HIV-1-infected monocyte-derived machrophages (MDM). MDM (A) and HIV-1ADA infected MDM (B) are cultivated in 6-well plates. Cytopreparations of MDM (C) and HIV-1ADA infected MDM (D) were immunostained with antibodies against HIV-1 p24. Virus-positive cells are black, original magnification x20. Arrows show multinucleated virus infected cells.

3.3.2.     Isolation of HIV From Infected Cerebrospinal Fluid (CSF)

1.            Monocytes cultured in a 6-well plate are obtained as described in Subheading

3.2.

2.            On d 7, add 1 mL of diluted patient CSF/well for infection or directly inoculate 100 pL of fluid into 1 mL total culture fluid volume.

3.            Incubate the cells at 37°C with 5% CO2 and 95% humidity for 24 h.

4.            After 24 h incubation add an additional 4 mL of fresh monocyte medium without MCSF.

5.            Every other day, collect the samples by replacing one half of culture media (per Subheading

3.3.1.). Samples are tested for RT activity and stored as described.

3.3.3.     Isolation of HIV From Infected Brain Tissue

1.            Prepare monocyte culture for infection as described in Subheading 3.2. On d 7, cocultivation of infected brain tissue and MDM is initiated.

2.            HIV-infected brain tissue obtained by autopsy is processed in a biological safety cabinet following at least BSL-2 practices. Basal ganglia or frontal cortex are optimal brain regions for viral rescue. Virus recovery is optimal if tissue is processed immediately after autopsy. Brain tissue can be frozen but freeze-thaw cycles diminish viral yields.

3.            Wear cut-resistant Kevlar gloves during tissue slicing.

4.            Transfer tissue into a sterile Petri dish, then wash and remove excess blood with PBS. Using scalpels, chop the tissue into 2-3-cm2 pieces. Remove any excess blood vessels and connective tissue with forceps.

5.            Remove the PBS and cover the tissue pieces with monocyte medium without MCSF. Slice the tissue into 1-2-mm2 pieces using scalpels.

6.            Aspirate medium from the monocyte culture and transfer equivalent amount of media containing the brain pieces. No more than 30-40% of the monolayer should be covered with the tissue pieces.

7.            Incubate the culture over night at 37°C with 5% CO2 and 95% humidity.

8.            The following day (d 8), remove tissue pieces along with the medium using a pipet without disturbing the cells. Replace with the equal amounts of monocyte media without MCSF.

9.            From d 10, do half-exchange and collect samples every 2 d as described in Subheading 3.3.1. Pool the samples that contain peak RT activity and store (Subheading 3.3.1.).

3.4.        RT Assay

1.            Pipet 10 pL of solution A into an appropriate number of wells in a 96-well round-bottom plate. Assay in quadruplicate. A blank (media), standards, and unknown samples are included in the assay. Use different dilutions of the standard, ranging from 0.1 to 10 U/10 pL. Dilute the standards immediately before use.

2.            Add 10 pL of each sample to appropriate wells and mix by swirling the plate.

3.            Incubate the plate for 15 min at 37°C with 95% humidity.

4.            Add 25 pL of solution B to each well. Solution B contains radioactive thymidine, appropriate cautions need to be taken while handling the radioactive material.

5.            Incubate the reaction plate for 18-24 h at 37°C with 95% humidity.

6.            After the incubation add 50 pL of ice-cold 10% TCA to each well and keep the plate at 4°C for at least 15 min.

7.            Collect the precipitate by harvesting the plate using a cell harvester.

8.            Place the filter paper disks separately into vials containing the scintillation fluid and count the radioactivity.

9.            Calculate the amount of RT activity in the unknown samples from the standard curve made by using different dilutions of commercial standard RT.

3.5.        Screening for Endotoxin and Mycoplasma

Endotoxin and mycoplasma contaminations are serious problems associated with cell culture (see Note 14). If they are left undetected, it is hard to conclude a negative result affected by these contaminations. HIV propagated on human cells, cell culture supernatants, and any homemade cell culture reagent are routinely tested for endotoxin and mycoplasma. All other cell culture reagents procured directly from the companies undergo equivalent quality control measures before being supplied.

3.5.1.     Endotoxin Assays

1.            Use pyrotubes for dilution of standards and the assay. The procedure must be performed in a biological safety cabinet (see Note 15).

2.            Prepare the various dilutions of CSE in reagent water: 100 pg/mL, 50 pg/mL, 25 pg/mL, and 12.5 pg/mL.

3.            Transfer 100 pL of each dilution into fresh pyrotubes for the assay, to prepare the standard curve. Use 100 pL of reagent water for the blank.

4.            Make at least two dilutions of the sample to be tested and take 100 pL of undiluted and diluted samples for the assay.

5.            Reconstitute LAL with 2 mL of reagent water (see Note 16). Add 100 pL of LAL reagent to the assay tubes with the samples. Vortex the tube immediately after adding LAL. Once the tube is set back in the test-tube rack, do not move the tube.

6.            After adding LAL to all the tubes, incubate at 37°C for 60 min. Any disturbance to the tubes during the incubation will cause false negatives.

7.            After 60 min observe each tube carefully for the gel clot. Take the tubes out of the rack one by one with minimal disturbance for observation.

8.            A positive (+) reaction appears as a gel clot. A positive/negative (±) reaction appears as a gel that breaks, or turbid precipitate. A negative (-) reaction has no gel formation.

3.5.2.     Mycoplasma

1.            Take 2 mL of the sample to be tested for mycoplasma detection assay.

2.            HIV must be inactivated before the assay. For inactivation, add 3 pL of tribatylphosphate and 20 pL of Triton X-100 (to get a final concentration of 1% Triton X-100) to 2 mL of virus and incubate at 37°C overnight.

3.            The following day, perform mycoplasma detection according to the kit manufacturer’s instructions (see Note 17).

4.            Notes

1.            Cell isolation and cell culture procedures must be carried out strictly under sterile conditions in a BSC. All pipets, centrifuge tubes, and tips must be sterile, and the packages should be opened only inside the BSC. The working area of the BSC should be wiped with 75% ethanol for sterilization.

2.            Do not insert the tubing directly into either PBS or cell suspension. Connect a sterile aspirating pipet to the tubing and insert the pipet into the reservoir. Do not let the reservoirs become empty while the pump is on, as air bubbles can enter the system. Connect another sterile aspirating pipet to the output tubing and put it in the collection bottle.

3.            Before and after using the elutriation system, it is very important to take proper precautions to clean and maintain the system to avoid clogging and to maintain sterile conditions.

4.            The flow rate, rotor speed to elute a particular cell type, is determined by the nomogram given in the instruction manual provided with the elutriation system. In the protocol, the rotor speed is mentioned in rpm as obtained from the nomogram to retain particles of 6 pm or larger at a flow rate of 40 mL/min.

5.            Cell analysis should be done fast without time delay as the pump cannot be stopped during elutriation. Otherwise, there can be loss of cells.

6.            Removing adhered monocytes either by cell scraping or trypsinization is not recommended for reculture, as the physiology of monocytes can get affected.

7.            Monocytes are adherent cells, so the supernatants can be collected carefully without disturbing the cells. If there are any loose cells present, spin the supernatant at 400g for 5 min, collect the supernatant, and transfer the cells back into the corresponding well.

8.            Human monocytes require MCSF for differentiation. After 6-7 d into culture, the cells begin making their own MCSF, and supplementation is no longer required (hence the change to monocyte medium without MCSF).

9.            Incubating the cells with HIV in a minimal volume for the first 4-5 h facilitates maximum HIV-cell interaction to enhance infection levels.

10.          Monocytes cultured between 6-9 d can be used for HIV infection. HIV-mac-rophage infection is highly dependent on cellular differentiation state. Between d 6 and d 7 of culture, the cells are differentiated enough to obtain successful viral infection.

11.          Two consecutive cultures showing RT activity indicates successful viral infection.

12.          The infected cultures should be closely monitored. The supernatants can be collected as long as there is an intact monolayer without complete loss of cells. An active cell culture is also indicated by the pH change to an acidic side (yellow) after 24 h of medium change.

13.          Storing viral aliquots in liquid nitrogen is recommended for long-term usage to retain the infectivity.

14.          Excellent tissue culture practices are extremely important, both for the safety of the personnel and for preventing endotoxin contamination. Human sweat, dust, and the like, are potential sources of endotoxin.

15.          Throughout the assay, it is important to note that improperly performed procedures can lead to false negatives, and that false positives can occur as a result of the introduction of endotoxin during the assay. Sterility and careful handling of reagents is crucial.

16.          Do not make up LAL in advance. Reconstituted LAL left to sit can cause both false-negatives and false-positives.

17.          Samples infected with mycoplasma will give a final ratio greater than 1, as detected by the luminometer.

References

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7 Persidsky, Y. and Gendelman, H. E. (2003) Mononuclear phagocyte immunity and the neuropathogenesis of HIV-1 infection. J. Leukoc. Biol. 74, 691-701.

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