关于流式抗体的制备，一些基本的程序（来自于BD公司）

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Procedure, v! f' c" M% L" j" p
1. Dilute unconjugated or biotin-conjugated monoclonal antibody by adding 20 of μL antibody to 30 μL of wash buffer for each test. Add the 50 μL of diluted antibody to microtiter wells or 12 x 75-mm tubes.
0 d: c, v; G! o6 K6 b2. Adjust concentration of the cell suspension to 2 x 107 cells/mL of wash buffer. Cells should be >90% viable.: a# S. {9 t5 |' {4 f" p) T4 }
3. Add 50 μL of the cell suspension (1 x 106 cells) to each microtiter plate well or to each 12 x 75-mm tube.
% s% o, j, B( g6 C' U4. For staining in microtiter plates: Incubate the mixture for 30 to 45 minutes on ice. Centrifuge at 200 x g for 3 minutes at 2° to 8°C. Remove the supernatant. Wash two times with 100 μL of cold wash buffer. Centrifuge after each washing at 200 x g for 3 minutes. Remove the supernatant.
. g) g2 w5 o, y# T" VFor staining in tubes: Incubate the mixture for 30 to 45 minutes on ice. Add 2 mL of cold wash buffer, and centrifuge at 300 x g for 5 minutes at 2° to 8°C. Remove the supernatant.8 v2 ^: b' t0 p8 d, l1 f/ _8 h) d
5. Dilute the appropriate second-step reagent (fluorochrome-conjugated avidin or goat anti-mouse Ig) in wash buffer according to the instructions provided on the appropriate second-step reagent data sheet.4 j1 M) J3 e: _7 t' a  v3 w, N
6. For staining in microtiter plates: Add the second-step mixture to the wells and incubate for 30 to 45 minutes on ice. Centrifuge at 200 x g for 3 minutes at 2° to 8°C. Remove the supernatant. Wash two times with 100 μL of cold wash buffer. Centrifuge after each washing at 200 x g for 3 minutes. Remove the supernatant. Resuspend cells in 200 μL of 1% paraformaldehyde solution and transfer samples to 12 x 75-mm tubes containing 300 μL of 1% paraformaldehyde solution. - N% U6 j6 {0 l" }
For staining in tubes: Add the second-step mixture to the tubes and incubate for 30 to 45 minutes on ice. Add 2 mL of cold wash buffer. Centrifuge at 300 x g for 5 minutes at 2° to 8°C. Remove the supernatant. Resuspend cells in 0.5 mL of 1% paraformaldehyde solution to approximately 1 x 106 cells/mL.9 @# F9 x% n/ T9 z
7. Store at 2° to 8°C until analyzed.
  \/ X) O9 g0 ~% n! W0 X- ~  q2 R8. Analyze on a FACS brand flow cytometer. Mix samples thoroughly before acquisition.! B( e% Q! {. m
参考！！
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