Detection of Fcγ Receptor-Mediated Adhesion and Phagocytosis

Summary

Author: J.E. Colligan et al, Translator: Xuitao Cao et al, This experiment is from "Compendium Immunology Laboratory Guide".

Operation method

Detection of Fcγ receptor-mediated adhesion and phagocytosis

Move

Basic scheme FcY receptor-mediated phagocytosis Material

Primary macrophages or macrophage cell lines (Elements 6.1 and 8.5)

R P M I -5 complete medium (or other medium)

Sheep red blood cells (S R B C ), diluted with Alsever's solution I : I (V /V) (Appendix 1)

Saline: 0.9 % (m/V) NaCl

200 to 900 Ci/g N a 251CrO4 (30 000 Ci/m m o l , ICN Biomedicals or DuPont N E N ) , saline preparation

Polyclonal anti-S R B C antibody (e.g., Diamedix) or anti-FcyRI monoclonal IgG2a type antibody (i.e., conditioner, hybridoma S.S-I, A T C C )

ACK lysate

0.5% (m / V ) SDS solution

Stimulants (e.g., mouse IFN-cx/press; see Module 6.4 for other activators)

8-channel pipettes (e.g. Costar Octapette)

9 6-well flat-bottomed cell culture plates (Falcon or Costar)

7 Liquid Flash Meter and 7 Liquid Flash Tubes

15m l and 50m l conical bottom centrifuge tubes

IEC Centrifuge (Clinical)

Superclearance Collection System (S C S ; Skatron)

1 . Macrophage suspension was prepared and the concentration was adjusted to 2. 5 X ^IO6 cells/m l . The concentration was adjusted to 2.5 X IO6 cells/m l. The cell suspension was added to each well of a 96-well flat-bottomed culture plate with an 8-channel pipette. Three replicate wells were set up for each experimental group and control group. Place the cells in a cell culture incubator and incubate until the cells are walled (mouse peritoneal macrophages need more than 4 h, other poorly differentiated macrophages may need more time).
(mouse peritoneal macrophages need more than 4 h, other poorly differentiated macrophages may need a longer time).

2. Add appropriate stimulants to stimulate macrophage Fc y R expression. Type I interferon, such as IFN-a or IFN-p, lOOU/m l, can significantly up-regulate the expression of macrophage FCYR after 48 h. It can be used as a positive control. The group treated with medium alone was used as a negative control. The optimal concentration of different stimulants to regulate the expression of FcYR should be determined according to the pre-test.
3 . The 7 liquid flash tubes were labeled and numbered

Adhesion mediated by the auxiliary program Fcyr Material

Additional materials (see basic program for other materials)
150m mol/L iodoacetic acid (I A A; freshly prepared, stored away from light)

2 4-well flat-bottomed cell culture plates (Costar or Falcon)

Oscillator, plate type

1 . Prepare a suspension of giant contempt cells and adjust the concentration to 8 X ¹⁰⁵ cells/ml. Add 500ul of cell suspension to a 24-well plate. Set up at least 2 duplicate wells for each experimental group and control group. After the cells have attached to the wall in the cell culture incubator, add the stimulant (see basic protocol, steps 1 and 2).

2 . Prepare isotope-labeled and antibody-conditioned SRBC (see basic protocol, steps 4 to 7). Inhibit macrophage phagocytosis by adding 0.I ml of 50 m mol/L iodoacetic acid to 9.9 ml of S RBC suspension.

3 . Add IOOUl of SRBC supernatant to a 7-liquid flash tube containing SCS and read the cp m value (this value is 1/5 of the maximum macrophage adhesion cp m value).

4 . Aspirate the macrophage culture supernatant. Add 500^1 iodoacetic acid-treated SRBC suspension and incubate for I h or for an appropriate period of time (see basic protocol, step 10).

5 . Gently aspirate the unadhered SRBC suspension. Wash the cells by slowly adding 500ul of medium along the wall of the well with an I m l pipette to avoid blowing up macrophages. Repeat the washing 3 times to fully remove the unadhered SRBC.

Note: The washing of the second culture plate should be started after the washing of one plate is completely finished.

6. Add 500W of SD S at 0.5% concentration. Add 500W of 0.5 % SD S and shake the plate gently for about 10 min to lyse the macrophages.

7 . Label the 7-fluid flash tubes by number and add to the SDS filter paper. Pipette 400M1 Macrophage Lysate with a micron pipette, carefully add to the appropriate 7-flash tube, and check the cpm value with the 7-flash meter.

8. Calculate the mean value of each sample (see basic protocol, steps 16 to 17). When the results are expressed in terms of SRBC binding, the cp m value of 400 ul of lysate should be converted to the cp m value of 500 ul of total lysate.

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