Important:Please read through instructions before you begin.

Initial plate handling:

· Do not remove the plates from the foil bags until they are ready to be used. The foil bag is the primary moisture barrier. Once the plates are removed from the foil bags, the antibodies must be reconstituted.

· Before removing the foil seal be sure to centrifuge plates to pellet lyophilized Ab cakes and use caution when removing the foil seal. Please see Reconstituting the antibody section below for details.

· After the foil seal is removed and prior to reconstitution, avoid placing the plastic lid or any cover on the plates or resealing the plates with an adhesive-based plate seal. In each case, the resulting static can cause the lyophilized Ab cakes to dislodge and escape from wells.

· You may notice that not all lyophilized Ab cakes have the same physical appearance. This is expected and will not affect performance of the antibodies.

Sample preparation:

· Some cell surface markers are sensitive to enzymatic digestion. When possible use a non-enzymatic cell or tissue dissociation buffer to prepare cells for flow cytometry. For enzymatic dissociation of adherent cell lines, we recommend using BD™ Accutase™ Cell Detachment Solution (Cat. No. 561527).

· Ensure that cells are in a single cell suspension. A DNase treatment step can mitigate cell clumping.

· Some antibodies specific for cell surface markers can produce artifacts (false positives and negatives) when used to stain fixed cells. If fixation is necessary, then staining live cells with subsequent fixation prior to analysis can help reduce these artifacts.

Recommendations for staining with antibodies:

· We recommend evaluating background staining on the cells by titrating the secondary biotinylated antibodies and Alexa Fluor® 647 Streptavidin as the tertiary reagent before attempting a full screen. Excess secondary antibody and tertiary reagent has been provided. Based on the cell types tested, we recommend using the biotinylated anti-Rat, anti-Mouse and anti-Syrian Hamster Ig secondary antibodies at 1.25 μg/ml (100 μl per well) and the biotinylated anti-Armenian Hamster Ig secondary antibody at 0.6 μg/ml (100 μl per well).

· While the majority of the antibodies in the panel were raised using Rat hosts, some of the antibodies were raised using Syrian Hamster, Armenian Hamster, or Mouse hosts. To ensure that the appropriate species-specific, biotinylated secondary antibody is used with cells stained with the corresponding primary antibodies, refer to the following Table and to the color-coded Array Layout on pages 9 and 10 of this manual.

PlateSecondaryPlate Map Well ColorWellsSecondary Control Well

1 Rat Red All wells A1

2 Rat Red A1-A12, B1-B12, C1- C8, D1-D7 A1

2 Syrian Yellow H1-H4 H4

3 Armenian Green A1-A12, B1-B12, C1-C2, D1-D6* A1

3 Mouse Blue F1-F12, G1-G10, H1- H6 H6

· Check for any crossreactivity with the biotinylated secondary antibodies if you plan to treat cells with an activating or inhibitory antibody of your choice (eg, with soluble or plate-bound antibodies).

· The kit contains 27 Mouse anti-Mouse specificity antibodies (including five Ig isotypes) in Plate 3.The biotinylated polyclonal Goat anti-Mouse Ig secondary reagent will bind to cells of the mouse B cell lineage that express surface Ig. Therefore we recommend that you use appropriate counter stains to delineate cell types.For example, an anti-CD3 antibody conjugated to a fluorochrome other than Alexa Fluor® 647may be used to gate on T cells during analysis.

· We recommend wells containing only Biotinylated Secondary Ab with Alexa Fluor® 647 Streptavidin controls and wells with only Unstained Cells as controls in each experiment. Well A1 in Plates 1 and 2 can be used for the anti-Rat Ig Secondary Antibody control. Plate 2, Well H4 can be used for the anti-Syrian Hamster Ig Secondary Antibody control. Plate 3, Well A1 can be used for for the anti-Armenian Hamster Ig Secondary Antibody control. Plate 3, Well H6 can be used for the anti-Mouse Ig Secondary Antibody control. Any of the remaining buffer wells can be used as Unstained Cells Only controls.

· Additional antibodies not currently included on the BD Lyoplate Mouse Cell Surface Screening Panel may be added to the screen in any of the gray wells shown on Plate 2 and 3 Plate Maps.

· Mouse BD Fc Block™ - Some antibody preparations may bind via their Fc portions to Fc Receptor-bearing cells resulting in high nonspecific background staining. BD Pharmingen™ Purified Rat anti-Mouse CD16/CD32 (Mouse BD Fc Block™)(Cat. No. 553141 or 553142) can be used to block the Fc-mediated adherence of antibodies to Mouse Fc Receptors. However, since the BD Fc Block antibody has a Rat IgG2b isotype, it can be used only with primary antibodies raised in Syrian Hamster, Armenian Hamster or Mouse hosts in Plates 2 and 3. Please Note: When using BD Fc Block, secondary anti-Ig antibodies that do not cross react with its Rat IgG2b isotype must be chosen..

· Armenian Hamster Ig isotypes can be used as controls for the 3 antibodies raised in Syrian Hamsters, eg, Ham IgG2, λ in Plate 3, Well D4 can be used as an Ig isotype control for the anti-CD28 antibody in Well H1, Plate 2. To use this Ig isotype for the Syrian Hamster anti-Mouse CD28 antibody, add the anti-Syrian Hamster Ig second step along with the anti-Armenian Hamster Ig second step in Step 14 below.

Flow cytometric analysis:

· For flow cytometric analysis we recommend running 500,000 to 1,000,000 cells per well for best results. However, we have been successful in running as few as 250,000 cells per well.

· For flow cytometric analysis we recommend using a 96-well High Throughput Screening (HTS) Plate Loader. If a plate loader is not available, transfer stained cells from 96-well plates into BD Falcon™ 12 x 75 mm round bottom tubes (Cat. No. 352008) for manual loading.

Reconstituting the antibodies:

· After removing BD Lyoplate™ Mouse Cell Surface Marker Screening Panel plates from foil bags, centrifuge at 300g for 5 minutes.

· Hold the plate firmly on the work bench and gently remove the foil seal starting from one end and pulling across the plate to completely remove the seal. Once the foil seal is removed, all lyophilized antibodies must be immediately reconstituted. Do not replace the lid on the plate prior to reconstitution.

· Using a multi-channel pipette, reconstitute lyophilized antibodies in 110 μl of 1X sterile filtered (0.2 µm pore size) PBS. This results in an antibody solution that contains five tests (20 μl/test). Be sure to use fresh pipette tips for each row to prevent well-to-well contamination. Allow antibodies to reconstitute for five minutes at room temperature.

· Store the reconstituted antibodies at 4°C until the cells are prepared for experiments. Reconstituted antibodies can be stored in plates with lids at 4°C for at least 10 days.

· Seal the plate edges (with lid on) with Parafilm® M laboratory film to prevent loss of reconstituted antibody solution due to evaporation.

Screening cells by flow cytometry: (Please see Step 6: ~300 ml of BD Pharmingen™ Stain Buffer (FBS) is needed for screening)

1. Prepare a single cell suspension of live cells from a cell line, tissue or a three dimensional culture. For adherent cell lines, we recommend using either a mild enzyme such as Accutase™ (Cat. No. 561527) or a non-enzymatic dissociation buffer.

2. Wash the cells in two to four volumes of 1X PBS. Centrifuge at 300g for 5 minutes.

3. Remove any cell clumps by passing the cells through a BD Falcon™ 40 or 70 μm cell strainer (Cat. No. 352340 or Cat. No. 352350).

4. Determine the cell concentration and total number of cells. If you are dissociating tissues or a three dimensional culture, we recommend treating the single cell suspension with DNase to mitigate cell clumping. Resuspend cells in the recommended growth media or 1X PBS with calcium and magnesium and 100 units of DNase/ml at 10 million cells per ml. Incubate for 15 minutes at room temperature.

5. Wash the cells in two to four volumes of 1X PBS. Centrifuge at 300g for 5 minutes.

6. You will need around 300 ml of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) for subsequent steps.

7. Resuspend the cells from Step 5 in BD Pharmingen Stain Buffer. You will need 110 to 220 million cells (in approximately 22 ml total volume) to fill the antibody-containing wells and the control wells (500,000-1,000,000 cells per well) of the three plates. The minimum number of cells per well will depend on the cytometer and/or loss of cells during washing. We have been successful in running as few as 250,000 cells per well.

8. Label three BD Falcon™ round bottom 96-well plates (Cat. No. 351177) as Plates 1, 2 and 3 for your sample plates.

9. Using a multi-channel pipette aliquot 100 μl of cell suspension to required wells of the three labeled round-bottom 96-well plates.

a. If you have a limited number of cells, you can omit Buffer Only control wells from Plates 2 and 3. Please refer to the Plate 2 and 3 Maps to identify wells that can be excluded taking into consideration Unstained Cells and Secondary Antibody controls.

10. Using a multi-channel pipette, pipette up and down 2-3 times to fully mix the reconstituted antibody from the first row of wells from the BD Lyoplate Screening Panel Plate 1. After mixing, add 20 μl of the antibody solution to the cells in the corresponding Sample Plate 1 wells. Change pipette tips. Continue to add reconstituted antibody to the corresponding sample wells for all remaining wells of each sample plate. Use fresh tips for every well. Incubate on ice for 20-30 minutes.

11. To wash, add 100 μl of BD Pharmingen Stain Buffer to each well. Centrifuge at 300g for 5 minutes.

12. Remove supernatant carefully and wash cells with an additional 200 μl per well of BD Pharmingen Stain Buffer. Centrifuge at 300g for 5 minutes.

13. During the centrifugation step of the final wash, dilute the secondary antibodies in BD Pharmingen Stain Buffer according to the following table:

Secondary AbDilutionFinal Concentration (μg/ml) Volume of Diluted Secondary Ab (ml) Rat 1:400 1.25 15.0 Syrian 1:400 1.25 0.8 Armenian 1:800 0.60 4.0 Mouse 1:400 1.254.0

14. Remove supernatant and apply 100 μl of the appropriate biotinylated secondary antibody directly to cells in each well containing primary antibody as shown in the Array Layout Plate Maps (pages 9 and 10). Also, select an additional well as a Secondary plus Tertiary Reagent control for each of the4 secondary antibodies. Use the table below for reference. Use remaining wells in Sample Plate 2 or 3 that do not contain antibody (gray colored plate map wells) to set up Unstained Cells Only controls.

PlateSecondaryPlate Map Well ColorWellsSecondary Control Well

1 Rat Red All wells A1

2 Rat Red A1-A12, B1-B12, C1- C8, D1-D7 A1

2 Syrian Yellow H1-H4 H4

3 Armenian Green A1-A12, B1-B12, C1-C2, D1-D6* A1

3 Mouse Blue F1-F12, G1-G10, H1- H6 H6

* If you want to use Armenian Hamster Ig Isotype controls for the 3 Syrian Hamster antibodies, add the biotinylated anti-Syrian Hamster secondary antibody along with the biotinylated anti-Armenian Hamster secondary antibody to wells D1 (Arm IgG1, κ), D3 (Arm IgG2, κ) and D4 (Arm IgG2, λ).

15. Incubate for 20-30 minutes on ice in the dark.

16. To wash, add 100 μl of BD Pharmingen Stain Buffer to each well. Centrifuge at 300g for 5 minutes.

17. Remove supernatant and wash cells with an additional 200 μl of BD Pharmingen Stain Buffer. Centrifuge at 300g for 5 minutes.

18. Remove supernatant and add 100 μl of Alexa Fluor® 647 Streptavidin to all wells containing cells stained with the biotinylated secondary antibodies (not to the wells selected as Unstained Cell controls). Dilute the Alexa Fluor® 647 Streptavidin 1:4,000 (0.5 μg/ml) in 22 ml of BD Pharmingen Stain Buffer.

19. Incubate for 20-30 minutes on ice in the dark.

20. To wash, add 100 μl of BD Pharmingen Stain Buffer to each well. Centrifuge at 300g for 5 minutes.

21. Remove supernatant and wash cells with an additional 200 μl of BD Pharmingen Stain Buffer. Centrifuge at 300g for 5 minutes.

22. At this point you may wish to fix your cells prior to analysis. To fix, remove supernatant and add 100 μl of 4% paraformaldehyde in 1X PBS or BD Cytofix™ Fixation Buffer (Cat. No. 554655) per well and incubate for 10 minutes. If the fixation step is not required, then go to step 24.

23. Wash cells twice with 1X PBS. Centrifuge at 300g for 5 minutes.

24. Remove supernatant and resuspend cells in 150 μl of BD Pharmingen Stain Buffer per well.

25. Analyze your samples using a flow cytometer. We recommend collecting at least 10,000 events per well. While the first plate is being read, store the other plates on ice in the dark.

Screening cells by bioimaging:

1. Seed the cells in appropriate culture medium at an appropriate cell density in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture cells to an appropriate density. We recommend 70-80% confluence for imaging screens.

2. Cell surface staining with antibodies from the BD Lyoplate should be performed on live cells as cellular fixation can cause artifacts (false positive and/or negative signals) with some cell surface markers. In cases where cells must be fixed prior to staining, we recommend confirming positive hits with a live cell sample stain using imaging or flow cytometry.

3. Using a multi-channel pipette add 20 μl of each reconstituted antibody to the corresponding wells of your sample plates and incubate on ice for 20-30 minutes. Stain cells directly in 50 to 100 μl of fresh growth media. If staining fixed cells, then stain cells in 1X PBS.

4. Wash cells twice in 100 μl 1X PBS.

5. Dilute the biotinylated secondary antibodies in growth media according to the following table:

Secondary AbDilutionFinal Concentration (μg/ml) Volume of Diluted Secondary Ab (ml) Rat 1:400 1.25 15.0 Syrian 1:400 1.25 0.8 Armenian 1:800 0.60 4.0 Mouse 1:400 1.254.0

6. Remove supernatant and apply 100 μl of the appropriate biotinylated secondary antibody directly to each well containing cells stained with the primary antibody as shown in the Plate Map. Also, select an additional well as a Secondary plus Tertiary Reagent control for each of the 4 secondary antibodies. Use the table below for reference. Use remaining wells in Sample Plate 3 that do not contain antibody (gray colored plate map wells) to set up Unstained Cell controls.

PlateSecondaryPlate Map Well ColorWellsSecondary Control Well

1 Rat Red All wells A1

2 Rat Red A1-A12, B1-B12, C1- C8, D1-D7 A1

2 Syrian Yellow H1-H4 H4

3 Armenian Green A1-A12, B1-B12, C1-C2, D1-D6 A1

3 Mouse Blue F1-F12, G1-G10, H1- H6 H6

7. Incubate for 20-30 minutes on ice in the dark.

8. Remove supernatant and wash cells twice in 100 μl 1X PBS.

9. Dilute the Alexa Fluor® 647 Streptavidin 1:4,000 (0. 5 µg/ml) in 22 ml of growth media. Add 100 μl of Alexa Fluor® 647 Streptavidin to all wells containing cells stained with the biotinylated secondary antibodies (not to the wells selected as Unstained Cells Only controls).

10. Incubate for 20-30 minutes on ice in the dark.

11. Remove supernatant and wash cells twice in 100 μl 1X PBS.

12. At this point you may wish to fix your cells prior to analysis. To fix, remove supernatant and add 100 μl of 4% paraformaldehyde in 1X PBS or BD Cytofix Fixation Buffer per well and incubate for 10 minutes. If you do not wish to fix your cells, then go to step 14.

13. Remove the fixative from the wells, and wash the well contents twice with 100 μl of 1X PBS.

14. Add 100 μl 1X PBS with a cell-permeable nucleic acid stain, such as Hoechst 33342 Solution (Cat. No. 561908).

15. Analyze your samples on a high content bioimager.

Suggested Companion Products

DescriptionSizeCatalog NumberBD Pharmingen™ Stain Buffer (FBS)500 ml554656BD Cytofix™ Fixation Buffer100 ml554655BD Accutase™100 ml561527BD Pharmingen™ Hoechst 33342 Solution1 mg/ml561908BD Pharmingen™ Fc Block0.5 mg/ml553141 or 553142

Related Products

DescriptionSizeCatalog No.BD Falcon™ 96-well Microplates, Black/Clear with Lid, High-Content Imaging Assays32/case353219BD Falcon 96-well Microplates, Round Bottom with Lid, Flow Cytometry Analysis50/case351177BD Falcon Round Bottom Tube, 12 x 75 mm1000/cs352008

Warnings and Precautions

The Mouse Cell Surface Marker Screening Panel (Part A), with Lyoplates 1, 2 and 3, contains sodium azide. Investigators should note that the following risk and safety statements are applicable:

Hazard symbols:

Harmful by inhalation

Xn Harmful

Hazard-determining components of labeling:

Sodium azide

Risk phrases:

Harmful in contact with skin

22 Harmful if swallowed

Safety phrases:

23 Do not breathe gas/fumes/vapour/spray

36 Wear suitable protective clothing

60 This material and its container must be disposed of as hazardous waste.